Bispecific antibody or antibody mixture with common light chains

ABSTRACT

The present application provides a bispecific antibody or an antibody mixture with common light chains and a preparation method therefor. the present application also provides a nucleic acid molecule encoding the antibody or the mixture, a recombinant vector and a recombinant cell comprising the nucleic acid molecule, as well as a detection and quantitation method for the antibody or the mixture.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.15/541,921, filed Jul. 6, 2017, which claims the benefit of and priorityunder 35 U.S.C. § 371 to Patent Cooperation Treaty applicationPCT/CN2016/070447, filed Jan. 8, 2016, which claims the benefit ofChinese Patent Application No. 201510008045.8, filed Jan. 8, 2015, theentire contents of which are incorporated herein by reference.

SEQUENCE LISTING

This application incorporates in its entirety the Sequence Listingentitled “20201023_0096-PA-001US.DIV1Sequence Listing as Filed_amended”(69,685 bytes), which was created on Oct. 22, 2020 and filedelectronically herewith.

TECHNICAL FIELD

The present invention relates to a bispecific antibody or an antibodymixture, and a preparation method of the bispecific antibody or theantibody mixture. The present invention also relates to a nucleic acidmolecule encoding the bispecific antibody or the antibody mixture, arecombinant vector and a recombinant cell containing the nucleic acidmolecule, and a detection and quantification method of the bispecificantibody or the antibody mixture.

BACKGROUND

The number of monoclonal antibody drugs being marketed rapidly increasesduring the recent 15 years, and has become a field of growth in thepharmaceutical industry. Since 1996, about 30 monoclonal antibody drugshave been approved in total, wherein the annual sale of 9 monoclonalantibody drugs is more than USD 1 billion. In 2010, the total sale ofthe monoclonal antibody drugs exceeds USD 30 billion, with an annualgrowth rate of more than 10%. Due to its strong target specificity, amonoclonal antibody can only inhibit a single target, while in manydiseases (including tumor and autoimmune diseases), it is needed toinhibit multiple signal pathways to avoid compensation effects. Forvirus infection diseases, due to high mutation rate of virus, it isoften needed to inhibit multiple antigen sites to prevent escape. Hence,there are several alternatives to solve this problem. One alternative isusing a polyclonal antibody, or a heterodimer (such as a bispecificantibody) obtained by modifying an Fc fragment of the antibody, so thatit will have activities against at least two different antigens or twodifferent binding sites of the same antigen. Another alternative isusing an antibody mixture, which may contain two or more antibodiesagainst different antigen epitopes on the same target or againstdifferent targets.

A bispecific antibody (BsAbs) is an immunoglobulin molecule containingtwo different ligand binding sites. It comprises two different Fabsequences, instead of the same sequences for the two Fab arms of aclassical antibody. Hence, two arms of the Y shaped antibody can bind todifferent antigen epitopes. Application of the bispecific antibody incancer treatment has been reviewed in multiple documents (Carter 2001;Chames and Baty 2009; Chames and Baty 2009). One arm of BsAbs can beconnected with a tumor cell surface related antigen and the other armcan trigger immune effector cells to further kill cells so as to killtumor cells with the help of the immune system.

For the preparation of a bispecific antibody, as early as in the 1990s,Carter et al. modified some amino acids of a heavy chain of an antibodyusing a “knob into hole” model to successfully achieve the preparationof the bispecific antibody (Ridgway, Presta et al. 1996; Carter 2001).However, in their research, the ability of preventing formation of “holeto hole” homodimers were still insufficient, about 5% of homodimers wasstill observed. After that, this research group tried to further improvethe content of heterodimers through methods such as randommutation-phage display, but the problem was not fundamentally solved.

The inventors of the present invention modified CH3 related amino acidsof Fc based on a charged amino acid interaction network to weakeninteractions of the domains between themselves (which facilitatesformation of homodimers) and enhance the interactions between differentdomains (which facilitates formation of heterodimers), therebysuccessfully solving the problem of 5% remaining homodimers in the “knobinto hole” model, and related methods have been described in a patentapplication (Publication No: CN102558355A).

In contrast to the heterodimer platform technology, development of anantibody mixture production platform is still in a relatively earlystage, wherein much attention has been drawn to an antibody mixturetechnology from the Danish company Symphogen A/S. This technology firstaims to obtain multiple antibodies against the same target by screeningusing an antibody screening platform, then constructing a cell strainfor each antibody respectively, then mixing seed solutions cultured indifferent flasks, and finally gradually amplifying the reaction andoptimizing the purification process to obtain a final product. Althoughby this method, multiple antibodies can be obtained directly from onerecombinant production process by culturing a mixed population ofmultiple cells, this scheme still has some potential problems due todifficulties in controlling mixed cell populations and complexitiesrelated with subsequent scale-up.

The applicant of the present invention invents a method for producing amixture comprising two or more homodimer proteins or antibodies in asingle recombinant cell through mutating Fc portions to change directinteractions between Fc fragments. This method avoids potentialdifficulties in process control and scale-up brought about by mixed cellculture, and provides a more economic and effective approach forantibody mixture preparation and production. This scheme has also beendescribed in an earlier patent application (Publication No:CN103388013A).

However, no matter which method discussed above is used, mismatchingbetween light chains and heavy chains can still happen when acomplete-antibody frame is utilized to prepare the bispecific antibodyor antibody mixtures thereof, thereby affecting the activity of theantibody. Currently, a relatively mature method in the art is Crossmabdeveloped by Roche (Genentech), that is, substituting light chain-heavychain sequences in one Fab to prevent mismatching between this lightchain sequence with light chain or heavy chain sequences of the otherFab (Patent Number US20090162359, US20120164726). Although this methodmay be used to solve most problems of heavy chain-light chainmismatching, yet new problems may be brought about due to modificationsin heavy chain and light chain sequences, for example, dissociation oflight chains, increase of multimers, and some influences on recognitionof antigen epitopes for some Fab sequences.

Herceptin (also referred to as Trastuzumab), as a first therapeuticmonoclonal antibody showing clinical effect in breast cancer, is ananti-human epidermal growth factor receptor 2(HER2) monoclonal antibody,which acts on HER2-Neu surface proteins of breast cancer cells tointerfere with a biological process of cancer cells and finally kill thecancer cells. The proper patient population for Herceptin is that ofbreast cancer with HER2 overexpression (immunohistochemical 3+ orfluorescence in situ hybridization FISH positive), and this groupapproximately accounts for 20-30% of all breast cancer patients.

Pertuzumab is a recombinant monoclonal antibody, which binds toextracellular domain II of HER-2 receptor to inhibit formation of dimersand inhibit a receptor-mediated signal transduction (Agus D B, GordonMs, Taylor C, et al. 2005). This may partially explain the reason forPertuzumab monoclonal antibody to inhibit growth of HER-2under-expression tumor, while Trastuzumab binds to an extracellular IVregion of the HER-2 receptor, and formation of dimers does not involvethe IV region. Hence, Trastuzumab is only effective for breast cancerpatients with HER-2 overexpression. At present, a phase II clinicaltrial for the treatment of terminal stage breast cancer with HER-2under-expression using Pertuzumab is undergoing. The research of Baselga(Baselga J, et al. 2007) et al. shows that the combination of Pertuzumaband Herceptin (Trastuzumab) has anti-tumor activity for HER-2 positivebreast cancer patients refractory to treatment. This result shows thatPertuzumab is efficacious for ⅕ of the patients (tumor is diminished ordisappeared), and the status of another ⅕ of the patients weremaintained stable for 6 months or longer. A result of a Phase IIIclinical trial for treating breast cancer with Pertuzumab shows thatthis drug can prolong progression-free survival of patients with ERBB2positive metastatic breast cancer.

Presently, Roche announced a latest trial result. The trial was a PhaseII therapy on the clinical effect for treating female breast cancerpatients positive for the protooncogene human epidermal growth factorreceptor 2 (HER2), using Pertuzumab and Herceptin (Trastuzumab) incombination with a chemotherapy (docetaxel). The data shown by theCancer Therapy & Research Center-American Association for CancerResearch (CTRC-AACR) in San Antonio Breast Cancer Seminar (SABCS)demonstrated that the complete remission rate (with a complete remissionrate of 45.8% of cases) of breast tumor treated by administrating acombination of the two antibodies and docetaxel in preoperativeneoadjuvant therapy is significantly enhanced by more than 50% ascompared to that of the combination of Herceptin and docetaxel (completeremission rate of 29.0% of cases). Comparing to Herceptin andchemotherapy, Pertuzumab and combination of Pertuzumab with docetaxelwould not cause side effects or significant increase of risks of heartdiseases.

The present invention takes Pertuzumab and Trastuzumab as examples toprepare a bispecific antibody and an antibody mixture having thefunctions of both Pertuzumab and Trastuzumab, and based on this,developed a new method for preparing a bispecific antibody or anantibody mixture, wherein light chains and heavy chains can assemblecorrectly.

SUMMARY

Through repeated experimentation, the inventors of the presentapplication surprisingly found that light chains originally present intwo antibodies or antibody mixtures can be replaced with a common lightchain so as to obtain a bispecific antibody or antibody mixturecomprising the common light chain. In the bispecific antibody orantibody mixture comprising the common light chain, the light chains andthe heavy chains are capable of assembling correctly. In addition, incomparison with the two original antibodies, the bispecific antibody hasgood binding activity, biological activity and stability, moreover, thebispecific antibody has better biological activity than the originalantibodies.

A first aspect of the present application relates to a bispecificantibody or an antigen binding portion thereof, wherein the bispecificantibody or the antigen binding portion thereof has a common lightchain, and wherein said common light chain refers to two light chainshaving the same sequence.

In one embodiment, heavy chains of the bispecific antibody or theantigen binding portion thereof are capable of correctly assembling withsaid light chains respectively under physiological conditions or duringin vitro protein expression.

In one embodiment, the common light chain of the bispecific antibody orthe antigen binding portion thereof is obtained by modifying twooriginal monoclonal antibodies (known monoclonal antibodies), and thesequence of the common light chain is different from that of a lightchain of at least one of the two original monoclonal antibodies. In oneembodiment, the common light chain is the same as the light chain of oneof the two original monoclonal antibodies, or is obtained viamodification on the basis of the two original monoclonal antibodies(such as amino acid sequence modification), and the objective of themodification is to maintain affinity with the respective antigen orantigen epitope as much as possible. In one embodiment, the modificationof amino acid sequence comprises mutation, deletion or addition of aminoacids, for example, the mutation, deletion or addition of no more than 3amino acids, preferably no more than 2 amino acids, and more preferablyno more than 1 amino acid.

In one embodiment, an Fc fragment of a heavy chain of the bispecificantibody or the antigen binding portion thereof is modified tofacilitate formation of a heterodimer protein.

In one embodiment, the two original monoclonal antibodies are Pertuzumaband Trastuzumab.

In one embodiment, the common light chain is capable of assembling witha heavy chain of Pertuzumab and a heavy chain of Trastuzumab,respectively.

In one embodiment, the common light chain is a light chain selected froma light chain of Pertuzumab or a light chain of trastuzumab, or a mutantthereof. In one embodiment, the heavy chains (comprising a variableregion and a constant region) of the bispecific antibody or the antigenbinding portion thereof can be the same as heavy chains of the twooriginal monoclonal antibodies, or be modified to facilitate formationof the heterodimer protein; the modification, for example, is amodification in the heavy chain Fc fragment so as to facilitateformation of the heterodimer protein.

In one embodiment, a sequence of a variable region of the common lightchain comprises a sequence selected from those as set forth in aminoacid positions 1-107 of SEQ ID NO: 1-SEQ ID NO: 6.

In one embodiment, a sequence of a light chain constant region comprisesa sequence selected from those as set forth in amino acid positions108-214 of SEQ ID NO: 1.

In one embodiment, heavy chain variable regions thereof are a heavychain variable region of Pertuzumab and a heavy chain variable region ofTrastuzumab, respectively.

In one embodiment, the heavy chain variable regions comprise sequencesas set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively.

In one embodiment, Fc fragments sequences of the heavy chain comprisesequences as set forth in SEQ ID NO: 25 and SEQ ID NO: 26, respectively.

In one embodiment, two heavy chains thereof comprise a sequence as setforth in SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

A second aspect of the present application relates to a mixture ofantibodies or antigen binding portions thereof, wherein said antibodiesor antigen binding portions thereof are capable of being producedcorrectly in one cell. The mixture comprises at least two antibodies orantigen binding portion thereof, and the antibodies or antigen bindingportion thereof have a common light chain, and the common light chainrefers to two light chain variable regions having the same sequence.

In one embodiment, heavy chains of the antibody or the antigen bindingportion thereof are capable of correctly assembling with the lightchains respectively under the physiological conditions or during invitro protein expression.

In one embodiment, the common light chain of the bispecific antibody orantigen binding portion thereof is obtained by modifying the twooriginal monoclonal antibodies (known monoclonal antibodies), thesequence of the common light chain is different from that of the lightchain of at least one of the two original monoclonal antibodies. In oneembodiment, the common light chain is the same as the light chain of oneof the two original monoclonal antibodies, or is obtained viamodification (such as amino acid modification) on the basis of the twooriginal monoclonal antibodies, the objective of the modification is tomaintain affinity with the respective antigen or antigen epitope as muchas possible. In one embodiment, the amino acid modification comprisesmutation, deletion or addition of amino acid, for example, mutation,deletion or addition of no more than 3 amino acids, preferably no morethan 2 amino acids, more preferably on more than 1 amino acid.

In one embodiment, the heavy chains of the bispecific antibody orantigen binding portion thereof are derived from the two originalmonoclonal antibodies, the sequence of the heavy chain variable regionand/or CH1 domain of the bispecific antibody or antigen binding portionthereof is the same as that of the original monoclonal antibodies.

In one embodiment, the heavy chain (including a variable region and aconstant region) of the bispecific antibody or antigen binding portionthereof may be the same as heavy chains of the two original monoclonalantibodies, or may be modified to facilitate formation of the homodimerprotein; the modification, for example, is a modification of heavy chainFc fragment to facilitate formation of the homodimer protein.

In one embodiment, the two original monoclonal antibodies are Pertuzumaband Trastuzumab.

In one embodiment, the common light chain is capable of assembling witha heavy chain of Pertuzumab and a heavy chain of Trastuzumab,respectively.

In one embodiment, the common light chain is selected from a light chainof Pertuzumab, a light chain of Trastuzumab or a mutant thereof.

In one embodiment, a sequence of a variable region of the common lightchain comprises a sequence selected from sequences as set forth in aminoacid positions 1-107 of SEQ ID NO: 1-SEQ ID NO: 6.

In one embodiment, a sequence of a constant region of the light chaincomprises a sequence selected from sequences as set forth in amino acidpositions 108-214 of SEQ ID NO: 1.

In one embodiment, heavy chain variable regions of the antibody orantigen binding portion thereof are a heavy chain variable region ofPertuzumab and a heavy chain variable region of Trastuzumab,respectively.

In one embodiment, a sequence of a variable region of the two heavychains comprises a sequence as set forth in in SEQ ID NO: 23 and SEQ IDNO: 24, respectively.

In one embodiment, Fc fragment sequences of the heavy chains of theantibody or antigen binding portion thereof comprise sequences as setforth in SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

In one embodiment, sequences of the heavy chains of the antibody orantigen binding portion thereof comprise sequences as set forth in SEQID NO: 21 and SEQ ID NO: 22, respectively.

A third aspect of the present application relates to a variant proteinof a HER2 protein extracellular domain, wherein, when comparing to asequence of a wild type HER2 protein extracellular domain, the variantprotein comprises mutations selected from the group consisting of:

1) a mutation of glutamic acid at position 558 and a mutation ofphenylalanine at position 573; and2) a mutation of serine at position 288 and a mutation of histidine atposition 296.

In one embodiment, a mutation from glutamic acid at position 558 toalanine.

In one embodiment, a mutation from phenylalanine at position 573 toalanine.

In one embodiment, a mutation from serine at position 288 to alanine.

In one embodiment, a mutation from histidine at position 296 to alanine.

In one embodiment, the HER2 variant protein comprises an amino acidsequence selected from the amino acid sequences as set forth in SEQ IDNO:13, SEQ ID NO: 14 and SEQ ID NO: 15.

In one embodiment, a sequence of the wild type HER2 proteinextracellular domain is as set forth in SEQ ID NO: 18.

A fourth aspect of the present application relates to a nucleic acidmolecule, encoding the bispecific antibody or antigen binding portionthereof according to any one of the first aspect of the presentapplication, or the bispecific antibodies or antigen binding portions inthe mixture according to any one of the second aspect of the presentapplication, or a part of the bispecific antibody or antigen bindingportion thereof (such as a light chain and/or a heavy chain), orencoding the HER2 variant protein according to any one of the thirdaspect of the present application.

A fifth aspect of the present application relates to a recombinantvector, comprising the nucleic acid molecule according to any one of thefourth aspect of the present application.

A sixth aspect of the present application relates to a recombinant cell,comprising the recombinant vector according to any one of the fifthaspect or the nucleic acid molecule according to any one of the fourthaspect of the present application.

A seventh aspect of the present application relates to a method forpreparing a bispecific antibody or antigen binding portion thereof basedon two monoclonal antibodies or antigen binding portions thereof againstdifferent antigen epitopes, comprising the following step of:

obtaining a sequence of a common light chain capable of assembling witha heavy chain of the two monoclonal antibodies respectively based onlight chain sequences of the two monoclonal antibodies, wherein saidcommon light chain refers to two light chains having the same sequence,preferably, the common light chain is a light chain of one of the twomonoclonal antibodies or a mutant of the light chain of one of the twomonoclonal antibodies.

In one embodiment, the method further comprises the following steps of:

constructing expression vectors comprising the common light chainsequence and the heavy chain sequence of the two monoclonal antibodies,respectively, to obtain two recombinant expression vectors; preferably,mutating the heavy chain sequences, especially the Fc fragment, tofacilitate aggregation of the Fc fragments of the two monoclonalantibodies having different heavy chains; and

introducing the two recombinant expression vectors into the same hostcell, and inducing expression to obtain the bispecific antibody or theantigen binding portion thereof.

In one embodiment, a variable region of the common light chain isobtained with the steps of: firstly determining interface amino acids inthe light chain variable region of the two monoclonal antibodiescontacting with their respective antigens or antigen epitopes, thendesignating the light chain variable region of one of the two monoclonalantibodies as a candidate common light chain variable region, comparingthe interface amino acids in said candidate common light chain variableregion with those in the light chain variable region of the other one ofthe two monoclonal antibodies when contacting with the antigen orantigen epitopes directed to by said one of the two monoclonalantibodies, to identify differential interface amino acids, andselecting a light chain variable region comprising fewer number of thedifferential interface amino acids as the common light chain variableregion; preferably, further mutating the common light chain variableregion to obtain a common light chain variable region having a betteraffinity to the corresponding antigen or antigen epitope. In oneembodiment, a method of obtaining a constant region of the common lightchain comprises the steps of: designating the light chain constantregion of the monoclonal antibody providing a variable region of thecommon light chain as a common light chain constant region, or furthermutating the light chain constant region to obtain a constant region ofthe common light chain.

In one embodiment, the mutation refers to modifications of the interfaceamino acids.

In one embodiment, better affinity with the antigen or antigen epitopemeans that the affinity between the common light chain and the twoantigens or antigen epitopes directed to by the bispecific antibody orantigen binding portion thereof are balanced so that the bispecificantibody or antigen binding portion thereof has better biologicalactivity and physicochemical properties (such as stability).

An eighth aspect of the present application relates to a method forpreparing a mixture comprising at least two monoclonal antibodies orantigen binding portions thereof, comprising the following steps of:

obtaining a common light chain sequence capable of respectivelyassembling with heavy chains of two monoclonal antibodies respectivelybased on light chain sequences of the two monoclonal antibodies, whereinthe common light chain refers to that the two light chains have the samesequence, preferably, the common light chain is a light chain of one ofsaid two monoclonal antibodies or a mutant of a light chain of one ofsaid two monoclonal antibodies.

In one embodiment, the method further comprises the following steps of:

constructing expression vectors comprising the common light chainsequence and the heavy chain sequence of the two monoclonal antibodies,respectively, to obtain two recombinant expression vectors; preferably,mutating the heavy chain sequences, especially the Fc fragment, tofacilitate aggregation of the Fc fragments of the monoclonal antibodieshaving the same heavy chains; and

introducing the two recombinant expression vectors into one host cell,and inducing expression to obtain a mixture of the antibodies or antigenbinding portions thereof.

In one embodiment, a method of obtaining a variable region of the commonlight chain is obtained with the steps of: firstly determining interfaceamino acids in the light chain variable region of the two monoclonalantibodies contacting with their respective antigens or antigenepitopes, then designating the light chain variable region of one of thetwo monoclonal antibodies as a candidate common light chain variableregion, comparing the interface amino acids in said candidate commonlight chain variable region with those in the light chain variableregion of the other one of the two monoclonal antibodies when contactingwith the antigen or antigen epitopes directed to by said one of the twomonoclonal antibodies, to identify differential interface amino acids,and selecting a light chain variable region comprising fewer number ofthe differential interface amino acids as the common light chainvariable region; preferably, further mutating the common light chainvariable region to obtain a common light chain variable region having abetter affinity to the corresponding antigen or antigen epitope. In oneembodiment, a method of obtaining a constant region of the common lightchain comprises the steps of: designating a light chain constant regionof the monoclonal antibody providing a variable region of the commonlight chain as a common light chain constant region, or further mutatingthe light chain constant region to obtain a constant region of thecommon light chain.

In one embodiment, the mutation refers to a mutation of the interfaceamino acids.

In one embodiment, better affinity with the antigen or antigen epitopemeans that the affinity between the common light chain and two antigensor antigen epitopes directed to by the two monoclonal antibodies in themixture are balanced so that the mixture has better biological activityand physicochemical properties (such as stability).

The present application also relates to a method for determining whetheror not an antibody or antigen binding portion thereof is a bispecificantibody or antigen binding portion thereof and/or a method forquantifying the same, comprising the following steps of (see the diagramin FIG. 25):

1) preparing a first specific antigen and a second specific antigenrespectively, wherein said first specific antigen is capable of bindingto a first antigen binding portion but not to a second antigen bindingportion in a bispecific antibody or an antigen binding portion thereof,and said second specific antigen is capable of binding to said secondantigen binding portion but not to said first antigen binding portion;2) coating an ELISA plate with the first specific antigen (or the secondspecific antigen), adding an antibody to be tested, after a period ofreaction, adding the second specific antigen comprising a label (or thefirst specific antigen comprising a label), after a period of reaction,adding a detection molecule capable of binding to the label, after aperiod of reaction, and obtaining results according to the detectionprinciple, thereby determining the reaction as positive or negative,wherein the detection molecule comprises a detectable label; and3) when the reaction is positive and the result is concentrationdependent, the antibody or antigen binding portion thereof is determinedto be a bispecific antibody or antigen binding portion thereof,optionally, further quantifying the bispecific antibody or antigenbinding portion thereof according to the obtained positive result.

In the present application, the first antigen binding portion and thesecond antigen binding portion respectively refers to two portions of abispecific antibody or antigen binding portion thereof assembling withdifferent antigens or antigen epitopes; in an embodiment, the firstantigen binding portion and the second antigen binding portion areobtained respectively via modification on the basis of the two originalantibodies, and the antigens or antigen epitopes directed to by thefirst antigen binding portion and the second antigen binding portion arethe same as those directed to by the two original antibodies,respectively.

In one embodiment, the first antigen and the second antigen are HERm1and HERm2.

In one embodiment, the labeled specific antigen is a specific antigenlabeled with biotin.

In one embodiment, the detection molecule is a substrate molecule usedfor detection, such as HRP-labeled streptavidin.

The present application also relates to a method for determining whetheror not a mixture of antibodies or antigen binding portions thereofcomprises a homodimer protein, the mixture comprises two antibodies (afirst antibody and a second antibody) or antigen binding portionsthereof, and the method comprises the following steps of (see thediagram in FIG. 26):

1) preparing a first specific antigen and a second specific antigenrespectively, wherein said first specific antigen is capable of bindingto the first antibody but not to the second antibody, and said secondspecific antigen is capable of binding to the second antibody but not tothe first antibody;2) coating an ELISA plate with the first specific antigen (or the secondspecific antigen), adding the mixture to be tested, after a period ofreaction, adding the first specific antigen comprising a label (or thesecond specific antigen comprising a label), after a period of reaction,adding a detection molecule capable of binding to the label, after aperiod of reaction, and obtaining results according to the detectionprinciple, thereby determining the reaction as positive or negative,wherein the detection molecule comprises a detectable label;3) separately, coating an ELISA plate with the first specific antigen(or the second specific antigen), adding the mixture to be tested, aftera period of reaction, adding the second specific antigen comprising alabel (or the first specific antigen comprising a label), after a periodof reaction, adding a detection molecule capable of binding to thelabel, after a period of reaction, and obtaining results according tothe detection principle, thereby determining the reaction as positive ornegative, wherein the detection molecule comprises a detectable label;4) when the reaction in the step 2) is positive and is concentrationdependent and the reaction in the step 3) is negative, the mixture isdetermined to comprise a homodimer protein and does not comprise anyheterodimer protein; when the reaction in the step 2) is positive andthe reaction in step 3) is positive, the mixture is determined tocomprise both homodimer protein and heterodimer protein.

In one embodiment, the first specific antigen and the second specificantigen are HERm1 and Herm2.

In one embodiment, the labeled specific antigen is an antigen labeledwith biotin.

In one embodiment, the detection molecule is a substrate molecule usedfor detection, such as HRP-labeled streptavidin.

The present application also relates to a composition (such as apharmaceutical composition), comprising the bispecific antibody orantigen binding portion thereof according to any one of the first aspectof the present application, and optionally pharmaceutically acceptablecarrier or excipient.

The present application also relates to a composition (such aspharmaceutical composition, comprising a mixture according to any one ofthe second aspect of the present application, and an optionalpharmaceutically acceptable carrier or excipient.

The present application also relates to a kit, comprising the bispecificantibody or antigen binding portion thereof according to any one of thefirst aspect of the present application, and optionally a buffer and/oran instruction.

In one embodiment, the kit is used for diagnosing HER2 positive tumor(such as breast cancer and gastric cancer).

The present application also relates to a kit, comprising a mixtureaccording to any one of the second aspect of the present application,and optionally a buffer and/or an instruction.

In one embodiment, the kit is used for diagnosing HER2 positive tumor(such as breast cancer and gastric cancer).

The present application also relates to use of the bispecific antibodyor antigen binding portion thereof according to any one of the firstaspect of the present application in the manufacture of a medicament forpreventing and/or treating HER2 positive tumor (such as breast cancerand gastric cancer).

The present application also relates to use of a mixture according toany one of the second aspect of the present application in themanufacture of a medicament for preventing and/or treating HER2 positivetumor (such as breast cancer and gastric cancer).

The present invention relates to use of the bispecific antibody orantigen binding portion thereof according to any one of the first aspectof the present application in the preparation of a reagent or a kit fordiagnosing HER2 positive tumor (such as breast cancer and gastriccancer).

The present application also relates to use of the mixture according toany one of the second aspect of the present application in thepreparation of a reagent or a kit for diagnosing HER2 positive tumor(such as breast cancer and gastric cancer).

The present application also relates to use of the variant protein ofHER2 protein extracellular domain according to any one of the thirdaspect of the present application in the detection of the bispecificantibody or antigen binding portion thereof according to any one of thefirst aspect or in the detection of the mixture according to any one ofthe second aspect of the present application.

The present application also relates to a method for preventing and/ortreating HER2 positive tumor (such as breast cancer and gastric cancer),comprising the step of administrating to a subject in need thereof aprevention or treatment effective amount of a bispecific antibody orantigen binding portion thereof according to any one of the first aspectof the present application.

The present application also relates to a method for preventing and/ortreating HER2 positive tumor (such as breast cancer and gastric cancer),comprising a step of administrating to a subject in need thereof aprevention or treatment effective amount of a mixture according to anyone of the second aspect of the present application.

The present application also relates to a method for diagnosing HER2positive tumor (such as breast cancer and gastric cancer), comprising astep of using the bispecific antibody or antigen binding portion thereofaccording to any one of the first aspect of the present application.

The present application also relates to a method for diagnosing HER2positive tumor (such as breast cancer and gastric cancer), comprising astep of using a mixture according to any one of the second aspect of thepresent application.

The present application also relates to a method for detecting thebispecific antibody or antigen binding portion thereof according to anyone of the first aspect of the present application or detecting themixture according to any one of the second aspect of the presentapplication, comprising a step of using the variant protein of the HER2protein extracellular domain according to any one of the third aspect ofthe present application.

The present application also relates to the bispecific antibody orantigen binding portion thereof according to any one of the first aspectof the present application for preventing and/or treating HER2 positivetumor (such as breast cancer and gastric cancer).

The present application also relates to the mixture according to any oneof the second aspect of the present application for preventing and/ortreating HER2 positive tumor (such as breast cancer and gastric cancer).

Inventions of the present application will be further described below.

In the present application, the term “antibody” refers to animmunoglobulin molecule consisting of two pairs of identical polypeptidechains (each pair has one “light” (L) chain and one “heavy” (H) chain).A light chain of an antibody can be a κ light chain or a λ light chain.A heavy chain can be a μ, δ, γ, α or ε heavy chain, and the antibodyisotype is correspondingly defined as IgM, IgD, IgG IgA and IgE. Withinthe light chains and the heavy chains, variable regions and constantregions are connected through “J” regions which consist of about 12 ormore amino acids, the heavy chains also comprise “D” regions whichconsist of about 3 or more amino acids. Each heavy chain consists of aheavy chain variable region (VH) and a heavy chain constant region (CH).The heavy chain constant region consists of 3 domains (CH1, CH2 andCH3). Each light chain consists of a light chain variable region (VL)and a light chain constant region (CL). The light chain constant regionconsists of one domain CL. The constant regions of the antibody canmediate binding of an immunoglobulin with a host cell or a factor,including binding of various cells (such as effector cells) of theimmune system with a first component (C1q) of a classical complementsystem. VH and VL regions can also be subdivided into highly variableregions (named complementary determining regions (CDR)), and in betweenthe CDRs, conservative regions known as framework regions (FR) aredistributed. Each VH and each VL consists of 3 CDRs and 4 FRs arrangedfrom amino terminals to carboxyl terminals in a sequence of FR1, CDR1,FR2, CDR2, FR3, CDR3 and FR4. Variable regions (VH and VL) of each heavychain/light chain pair respectively form antibody binding portions.Distribution of amino acids in various regions or domains follows thedefinition in Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk (1987) J. Mol. Biol. 196:901-917, or Chothia et al (1989)Nature 342:878-883. The term “antibody” is not limited by any specificmethod for producing an antibody. For example, it comprises,particularly, a recombinant antibody, a monoclonal antibody and apolyclonal antibody. The antibodies can be antibodies of variousisotypes, for example, IgG (such as IgG1, IgG2, IgG3 or IgG4 subtype),IgA1, IgA2, IgD, IgE or IgM antibody.

In the present application, the term “antigen binding portion” of theantibody refers to one or more portions of a full-length antibody, theantigen binding portion maintains the ability of binding to an antigen(such as HER2) that is the same as that bound by the antibody, andcompetes with the full-length antibody for the specific binding to anantigen. General reference is made to Fundamental Immunology, Ch. 7(Paul, W., ed., edition II, Raven Press, N.Y (1989), which isincorporated herein by reference in its entirety and for all purposes.The antigen binding portion can be produced using a recombinant DNAtechnology or through enzymatic or chemical breakage of a full-lengthantibody. In some cases, the antigen binding portion comprises apolypeptide such as a Fab, a Fab′, a F(ab′)2, a Fd, a Fv, a dAb, acomplementary determining region (CDR) fragment, a single-chain antibody(such as a scFv), a chimeric antibody, and a diabody, and it comprisesat least the part of the antibody sufficiently endowing the polypeptidewith the specific antigen binding ability. The antigen binding portion(such as the above antibody fragment) of the antibody may be obtainedfrom a given antibody (such as monoclonal antibody 2E12) using aconventional technology (such as recombinant DNA technology or enzymaticor chemical breakage process) known to those skilled in the art, and arescreened for its specificity in a process that is the same for screeningfull-length antibodies.

In the present application, the term “Fd fragment” refers to an antibodyfragment consisting of V_(H) and C_(H)1 domains; the term “Fv fragment”refers to an antibody fragment consisting of V_(L) and V_(H) domains ofa single arm of an antibody; the term “dAb fragment” refers to anantibody fragment consisting of a V_(H) domain (Ward et al, Nature341:544-546 (1989)); the term “Fab fragment” refers to an antibodyconsisting of V_(L), V_(H), C_(L) and C_(H)1 domains; and the term“F(ab′)2 fragment” refers to an antibody fragment comprising two Fabfragments connected through a disulfide bridge in a hinge region.

In the present application, the term “antibody Fc fragment” is a termknown to those skilled in the art and is defined based on proteolysis ofan antibody by papain, and refers to a human immunoglobulin chainconstant region, especially a carboxyl terminal of an immunoglobulinheavy chain constant region or a part thereof. For example, animmunoglobulin Fc region may comprise combinations of two or moredomains selected from heavy chain CH2, CH3 and CH4 with animmunoglobulin hinge region. Based on amino acid sequences of the heavychain constant region, immunoglobulins may be divided into differenttypes, mainly the following five types: IgA, IgD, IgE, IgG and IgM, someof which can be further divided into subtypes (isotypes), for example,IgG-1, IgG-2, IgG-3, IgG-4, IgA-1 and IgA-2. Selecting specificimmunoglobulin Fc regions from specific types and subtypes of theimmunoglobulin is within the knowledge of those skilled in the art.

In one embodiment, the antibody Fc fragment used in the presentapplication comprises at least one immunoglobulin hinge region, one CH2domain and one CH3 domain, for example, a human IgG1 Fc.

In the present application, the term “bispecific antibody” refers to anantibody that can respectively bind with two antigens or antigenepitopes, comprising a light chain and a heavy chain of an antibodycapable of specifically binding to a first antigen or antigen epitope,and a light chain and a heavy chain of an antibody capable ofspecifically binding to a second antigen or antigen epitope. In oneembodiment, in the bispecific antibody, the antibody light chain capableof specifically binding to a first antigen or antigen epitope and theantibody light chain capable of specifically binding to a second antigenor antigen epitope have the same sequences. In one embodiment, in thebispecific antibody, the antibody heavy chain capable of specificallybinding to a first antigen or antigen epitope and the antibody heavychain capable of specifically binding to a second antigen or antigenepitope have different sequences.

In the present application, the term “epitope” or “antigen epitope”refers to a portion in an antigen specifically bound by animmunoglobulin or an antibody. “Epitope” is also known as “antigenicdeterminant”. The “epitope” or “antigenic determinant” often consists ofchemical reactive surface groups of a molecule, such as amino acid orcarbohydrate or glycosyl side chains, and often has specificthree-dimensional structure features and specific charge features. Forexample, the epitope, which can be “linear” or “conformational”, oftencomprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15continuous or non-continuous amino acids in a unique spatialconformation. See the reference, for example, Epitope Mapping Protocolsin Methods in Molecular Biology, volume 66, G. E. Morris, Ed. (1996). Ina linear epitope, all the interaction sites between a protein and amolecule that it interacts with (such as an antibody) are arrangedlinearly along the primary amino acid sequence of the protein. In aconformational epitope, interaction sites are arranged across theprotein at discrete sites separate from each other.

In the present application, 20 conventional amino acids andabbreviations thereof comply with conventional rules. Reference may bemade to, Immunology—A Synthesis (Edition II, E. S. Golub and D. R. Gren,Eds., Sinauer Associates, Sunderland, Mass. (1991)), which isincorporated herein by reference.

In the present application, light chain sequences (especially variableregion sequences) of two monoclonal antibodies (namely originalantibodies) against different antigens or antigen epitopes are analyzedand verified to obtain a common light chain capable of assembling withthe heavy chains of the two monoclonal antibodies. After assembling withthe heavy chains, the common light chain still can specifically bind tothe antigens or antigen epitopes directed to by the original monoclonalantibodies.

In the present application, the common light chain can be used forexpressing the bispecific antibody, and can also be used for expressinga mixture comprising two antibodies; when the bispecific antibody isexpressed, the antibody comprises a light chain and a heavy chain whichare capable of binding to a first antigen, and a light chain and a heavychain which are capable of binding to a second antigen, wherein thesequences of the two light chain are completely the same, namely, is acommon light chain; and when the antibody mixture is expressed, eachantibody respectively comprises two light chains and heavy chains,wherein sequences of the light chains are completely the same, namely,is the common light chain.

In the present application, light chain constant regions of the twooriginal antibodies can be of κ type or λ type; the κ-type light chainconstant region comprises various allotypes, such as Km1, Km2 and Km3;the λ-type light chain constant region comprises various allotypes, suchas CL1, CL2, CL3, CL6 and CL7.

It is known in the art that the variable region is crucial for specificbinding between the antigen and the antibody, thus, during the processof modifying or obtaining an antibody, the selection and modification ofthe variable region sequences are critical. Hence, in the presentapplication, in order to obtain the bispecific antibody or the antibodymixture having the common light chain, the variable regions of thecommon light chain need to be obtained firstly. After selecting thelight chain variable region of one original monoclonal antibody or amutant thereof as the variable region of the common light chainaccording to the method discussed above, the constant region of thecommon light chain is determined. Normally, the common light chainconstant region is determined to be the light chain constant region ofthe monoclonal antibody from which the common light chain variableregion is derived. In some cases, the light chain constant region of theother monoclonal antibody may be determined to be the common light chainconstant region. When necessary, the original light chain constantregion can be modified (e.g., by addition, deletion or mutation of aminoacid, etc.) based on knowledge known in the art to obtain a moresuitable constant region of the common light chain, for example, aftermodification, the common light chain constant region has a better ADCC,CDC, endocytosis, stability, immunogenicity or half-life etc.

In the present application, the heavy chain type of the two originalantibodies can be the same or different, preferably, the heavy chainsare of the same type. In one embodiment, when preparing the bispecificantibody and the antibody mixture, the sequences of the variable regionand the CH1 domain of the heavy chain are unchanged comparing to that ofthe original antibodies.

In the present application, two arms of the bispecific antibody or theantibodies of the mixture containing two antibodies are both derivedfrom two original monoclonal antibodies. When preparing the bispecificantibody or the antibody mixture, only sequences of the light chainvariable region would be changed to obtain the common light chain, whilesequences of the heavy chain variable region do not need to be changed.In other words, in the bispecific antibody or the antibody mixtureprepared, sequences of the antibody heavy chain variable regions may bethe same as that of the original antibody, but sequences of at least onelight chain variable region shall be different from that of the originalantibody.

In the present application, the two original monoclonal antibodies canbe selected according to different demands or objectives, for example,the selected two monoclonal antibodies can be against different antigenepitopes of the same antigen; alternatively, one of selected antibodiesmay bind to a related antigen on the surface of a tumor cell while theother antibody may trigger an immunologic effector cell so as to furtherkill a cell.

In the present application, when preparing the bispecific antibody, theheavy chain (such as an Fc fragment) can be modified based ontechnologies known in the art to facilitate formation of the heterodimerprotein during antibody expression.

In the present application, when preparing the antibody mixture, theheavy chain (such as an Fc fragment), can be modified based ontechnologies known in the art so as to facilitate formation of thehomodimer protein during antibody expression.

In the present application, technologies for modifying an Fc fragment ofthe antibody heavy chain to facilitate formation of the homodimerprotein or the heterodimer protein are known in the art, for example,reference may be made to, Ridgway, Presta et al. 1996, Carter 2001,Patent CN 102558355A and Patent CN 103388013A.

In the present application, technologies of fusing polypeptidesrecognizing different antigen epitopes include but is not limited to,for example, a heterodimer Fc fusing technology as shown in theexamples, it can also be an “Fab” technology, see FIG. 1.

In the present application, the heterodimer Fc fusing technology used inthe present application may be based on a “knob”-“hole” model, and itmay also be based on a “charge repulsion model”, but is not limited tothese two models.

In the present application, the platform capable of producing andpreparing antibody mixtures in a single recombinant cell as used in thepresent application may be based on the “charge repulsion” model, but isnot limited to this model.

In some embodiments of the present application, when preparing thebispecific antibody or the antibody mixture, the nucleic acid moleculeencodes a light chain and/or heavy chain of an antibody against a firstantigen, or encodes a light chain and/or heavy chain of an antibodyagainst a second antigen. In some embodiments of the presentapplication, the light chain is the common light chain; in someembodiments of the present application, the Fc fragment of the heavychain is modified.

In some embodiments of the present application, the vector can be acloning vector or an expression vector. The cloning vector is used forcloning a related fragment of an antibody; the expression vector is usedfor expressing a bispecific antibody or an antibody mixture. A vectorsuitable for antibody expression can be selected according to commonknowledge in the art. In some embodiments of the present application,the expression vector is pcDNA4m, which is obtained by modifying thevector pcDNA4/myc-HisA.

In some embodiments of the present application, the expression vectorcomprises a nucleic acid molecule encoding the light chain and/or heavychain of the antibody against the first antigen, or a nucleic acidmolecule encoding the light chain and/or heavy chain of the antibodyagainst the second antigen.

In some embodiments of the present application, the host cell is a hostcell suitable for the expression of the antibody, for example, aprokaryotic cell (such as E. coli) or a eukaryotic cell; the eukaryoticcell, for example, is a yeast cell, a plant cell or a mammal cell, andthe mammal cell, for example, is a CHO cell, HEK293 cell or a myelomacell, etc.

In some embodiments of the present application, the host cellsimultaneously comprises an expression vector which is capable ofexpressing the light chain and/or heavy chain of an antibody against thefirst antigen and an expression vector which is capable of expressingthe light chain and/or heavy chain of an antibody against the secondantigen; in some embodiments of the present invention, the light chainis the common light chain; in some embodiments of the presentapplication, the Fc fragment of the heavy chain is modified. When thehost cell is used to express a bispecific antibody, through amodification of the Fc fragment, the light chains and heavy chains ofantibodies against different antigens are more easily to assemble toform the bispecific antibody; when the host cell is used to express anantibody mixture, through a modification of the Fc fragment, lightchains and heavy chains of antibodies against the same antigen are moreeasily to assemble to form the antibody mixture.

The bispecific antibody or the antibody mixture can be purified from thehost cell using standard experiment approaches. Purification methodsinclude but are not limited to a chromatographic technique such asvolume exclusion, ion exchange, affinity chromatography andultrafiltration. In some embodiments of the present application, abispecific antibody and an antibody mixture are purified with ProteinAaffinity chromatography.

In the present application, the bispecific antibody or antigen bindingportion thereof or the mixture thereof can also be co-administered witha chemotherapeutic drug and/or other antibodies, thus a composition ofthe present application may also comprise a chemotherapeutic drug and/orother antibodies.

In the present application, the chemotherapeutic drug includes but isnot limited to Adriamycin, cyclophosphamide and taxane [Taxol andTaxotere], Xeloda, Gemzar, Navelbine, Tamoxifen, aromatase inhibitors(Arimidex, Felon and Arnoux), 5-FU & folinic acid, camptosar,Oxaliplatin, cis-plantinum, Paraplatin, Estramustin, Novantrone,Metacortandracin, Oncovin etc., or a combination thereof.

In the present application, by mutating a HER2 protein, a HER2 proteinmutant capable of only specifically binding to one of Pertuzumab andHerceptin is obtained. In some embodiments of the present application,the HER2 protein mutant is used to identify the bispecific antibody andthe antibody mixture.

In the present application, double-antigen sandwich ELISA (also known asbridge ELISA) is used in combination with the HER2 protein mutant todetermine whether or not the antibody is a bispecific antibody, orwhether or not the antibody mixture comprises a homodimer protein,further, to quantify the bispecific antibody or homodimer proteins inthe antibody mixture.

In the present application, the double-antigen sandwich ELISA method isknown in the art, and its principle is that an antigen bound to a solidsupport and an enzyme labeled antigen are used to respectively bind tothe two antigen binding sites of a candidate antibody molecule in asample, forming a solid phase antigen-antibody-enzyme labeled antigenimmune complex. The detection steps of this method, for example,comprise: (1) coating a solid support with a specific antigen,incubating for a period of time to form a solid support antigen, washingand removing unbound antigen and impurities; (2) adding a sample andincubating for the antibodies in the sample and the antigen on the solidsupport to interact sufficiently so as to form a solid supportantigen-antibody complex, and washing and removing other unboundsubstances; (3) adding an enzyme labeled antigen, incubating to form asolid support antigen-candidate antibody-enzyme labeled antigen sandwichcomplex, and washing and removing any unbound enzyme labeled antigen;(4) adding a substrate for color developing. Enzymes on the solidsupport will catalyze the substrate to produce a colored product, andthe quantity of the antibodies in the sample may be measured withcolorimetry.

In the present application, a HER2 positive tumor comprises a HER2protein over-expressed tumor (such as breast cancer, a gastric cancer,esophagus cancer, ovarian cancer, endometrial cancer, bladder cancer,lung cancer, colon cancer and head and neck neoplasm), and alsocomprises a HER2 protein under-expressed tumor (such as HER2under-expressed breast cancer and lung cancer).

In the present application, a common light chain capable of respectivelyassembling with heavy chains of two different antibodies can be obtainedthrough the analysis of sequences of the light chains of the twoantibodies, and a bispecific antibody and an antibody mixture having thecommon light chain are prepared accordingly. Experimental resultsdemonstrated that the bispecific antibody and the antibody mixtureprepared by the method has good binding activity, biological activityand stability, and has better biological activity than the originalantibody.

The common light chain technology is simple and controllable, andeffectively solves the problem of mismatching between heavy chains andlight chains of bispecific antibodies, while the stability, activity andpurity of the bispecific antibodies are not affected. For antibodymixtures, the common light chain technology allows antibodies to beexpressed in the same host cell, which avoids the difficulties of mixedcell population culture and facilitate large-scale production.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of heterodimer protein fusion. Panel aillustrates a heterodimer Fc fusion technology, and Panel b illustratesa “Fab” technology.

FIG. 2 illustrates recognition of light chain hypervariable regions ofPertuzumab and Trastuzumab, wherein A demonstrates the recognitionresult of a light chain hypervariable region of Pertuzumab, Bdemonstrates the recognition result of a light chain hypervariableregion of Trastuzumab, and C demonstrates a comparison result ofPertuzumab and Trastuzumab light chains and shows a comprehensiveanalysis result of the antigen interface amino acid.

FIG. 3 is a structural diagram of a Trastuzumab Fab fragment and a Her2extracellular domain (ECD).

FIG. 4 illustrates an SDS-PAGE electrophoretic analysis result of Her2ml and Herm2 variant proteins (18% SDS-PAGE non-reduced condition). 1:HER2 m1; 2: HER2m2; M: protein MW standard.

FIG. 5 illustrates detection of specific binding of a HER2 variantprotein with Trastuzumab or Pertuzumab using ELISA.

FIG. 6 is a reduced SDS-PAGE (12% SDS-PAGE reduced condition) resultshowing primary detection of a common light chain monoclonal antibodyprotein sample obtained by one-step affinity chromatographicpurification. 1-6: TmabCLC1-6; 7-12: PmabCLC1-6; M: protein MW standard.

FIG. 7 illustrates affinity of Trastuzumab comprising the common lightchain to its specific antigen HER2m1.

FIG. 8 illustrates affinity of Pertuzumab comprising a common lightchain to its specific antigen HER2m2.

FIG. 9 is an SDS-PAGE (12% SDS-PAGE reduced condition) result showingprimary detection of a KN026 antibody protein sample obtained byone-step affinity chromatographic purification.

1: KN026 transient expression cell culture supernatant; 2: KN026affinity chromatography flow through; 3: purified protein sample(reduction) after KN026 one-step affinity chromatography; 4: purifiedprotein sample (non-reduced) after KN026 one-step affinitychromatography; M: protein MW standard.

FIG. 10 illustrates an SE-HPLC detection result of KN026 antibodyprotein purity.

FIG. 11 illustrates affinity curves showing recognition of two antigensby bispecific antibody KN026.

FIG. 12 is an SDS-PAGE (12% SDS-PAGE reduced condition) result showingprimary detection of a KN010 antibody protein sample obtained byone-step affinity chromatographic purification.

FIG. 13 illustrates an SE-HPLC detection result of mixed antibodyprotein KN026.

FIG. 14 illustrates affinity curves showing recognition of two antigensby the mixed antibody protein KN026.

FIG. 15 illustrates concentration dependent curves of binding of Ptmabbispecific antibody (KN026), Pertuzumab and Trastuzumab with BT474cells.

FIG. 16 illustrates concentration dependent curves of binding of Ptmabbispecific antibody (KN026), Pertuzumab and Trastuzumab with N-87 cells.

FIG. 17 illustrates concentration dependent curves of binding of Ptmaband Tmab antibody mixture (KN010), Pertuzumab and Trastuzumab with BT474cells.

FIG. 18 illustrates inhibition effects of KN026, Trastuzumab andTrastuzumab+Pertuzumab drug combination on human breast cancer BT474cell proliferation.

FIG. 19 illustrates inhibition effects of KN026, Trastuzumab andTrastuzumab+Pertuzumab drug combination on human gastric cancer N-87cell proliferation.

FIG. 20 illustrates thermal stability test results (Tm values) of aPtmab bispecific antibody KN026 (light curve) and a Trastuzumabreference sample (dark curve).

FIG. 21 illustrates pharmacokinetic curves of KN026 and Trastuzumab.

FIG. 22 illustrates influence of a Ptmab bispecific antibody on thetumor size of a human ovarian cancer SKOV3 nude mouse xenograft.

FIG. 23 illustrates influence of a Ptmab bispecific antibody on thetumor size of a human gastric cancer N-87 nude mouse xenograft.

FIG. 24 illustrates influence of a PTmab bispecific antibody on thetumor size of a human gastric cancer N-87 nude mouse xenograft.

FIG. 25 is a schematic diagram of a method for determining whether ornot an antibody is a bispecific antibody and a diagram of aquantification method.

FIG. 26 is a schematic diagram of a method for determining whether ornot an antibody mixture comprises a homodimer protein.

FIG. 27 illustrates dosage dependency of a PTmab bispecific antibody onan HER2 under-expressed human non-small cell lung cancer NCI-H522 mousexenograft model.

FIG. 28 illustrates that the pharmaceutical effect of a PTmab bispecificantibody on a HER2 under-expressed human non-small cell lung cancerNCI-H522 mouse xenograft model is comparable to that of administeringequal mole of Trastuzumab standard sample together with equal mole ofPertuzumab.

DETAILED DESCRIPTION

The embodiments of the present application will be described in detailin light of the examples, but it will be understood by those skilled inthe art that the examples below are only for illustrating the presentapplication, rather than for limiting the scope of the presentapplication. Where specific conditions are not indicated, theexperiments are usually carried out in accordance with conventionalconditions or conditions recommended by the manufacturer. Where sourcesof the reagents or devices used are not indicated, conventional productsavailable on the market were used.

Example 1 Obtainment of Common Light Chain 1. Obtainment of Sequencesand Structures

Complex crystal structures of Trastuzumab and Pertuzumab are obtainedfrom a protein data bank (PDB, www.pdb.org) respectively, the PDB numberof Trastuzumab is 1N8Z, and the PDB number of Pertuzumab is 1S78. Twoscreening strategies can be used to identify amino acid contacts betweenCH3-CH3: (i) distance of amino acid interaction and (ii) solventaccessibility region analysis. Here, the analysis was performedaccording to the distance of amino acid interaction.

2. Obtainment of Monoclonal Antibody Light Chain and Antigen HER2Interface Amino Acid

In accordance with the principles of amino acid contact, the interfaceamino acids refer to such amino acids: the distance between a heavy atomof a side chain of the amino acid and a heavy atom of any amino acidfrom another peptide chain is less than a threshold value. Here, thethreshold value was determined to be 4.5 Å. 5.5 Å can also be used insome cases (Bahar and Jernigan 1997). Table 1 is a list of amino acidsmediating contact between a Trastuzumab light chain and the antigenHER2. 12 interface amino acids of Trastuzumab are shown in the Table 1,which are selected according to the principles of amino acid contact.

TABLE 1 Trastuzumab light chain-antigen HER2 interface amino acid listTrastuzumab Light Chain Antigen HER2 ASP28A(CL1) GLU598C ASN30A(CL1)PRO571C, ASP596C, GLU598C, ALA600C, CYS601C, GLN602C THR31A(CL1)CYS601C, GLN602C, PRO603C ALA32A(CL1) PRO571C TYR49A PRO603C SER50A(CL2)LYS593C, PRO603C PHE53A(CL2) PRO603C, CYS604C, PRO605C ARG66A ASP596C,GLU598C HIS91A(CL3) ASP570C, PRO571C, PRO572C TYR92A(CL3) LYS569C,PRO71C, PRO572C, GLU598C, ALA600C THR93A(CL3) ASP560C, PRO572CTHR94A(CL3) ASP560C, PRO572C

Table 2 is a list of amino acids mediating contact between a Pertuzumablight chain and an antigen TER2. 8 interface amino acids of Pertuzumabare shown in the Table 2, which are selected according to the principlesof amino acid contact.

TABLE 2 Pertuzumab light chain and antigen HER2 interface amino acidlist Pertuzumab Light Chain Antigen HER2 ILE31C(CL1) SER313A LEU46CHIS296A TYR49C HIS296A, CYS312A, SER313A, LYS314A, PRO315A SER50C(CL2)SER313A TYR53C(CL2) SER313A, LYS314A, PRO315A TYR55C(CL2) LEU295A,HIS296A THR56C(CL2) LEU295A, HIS296A TYR94C(CL3) THR254A, ASP255A,THR256A, PHE257A

3. Identification of Pertuzumab and Trastuzumab Light Chain HyperviableRegions (CDRL1, CDRL2, CDRL3)

Pertuzumab and Trastuzumab light chains are identified usinghypervariable region recognition system-kabat numbering, and thesoftware can be found at http://www.bioinf.org.uk/abs/abnum/. Theidentification result for a Pertuzumab light chain hypervariable regionis shown in FIG. 2-A, and the identification result of a Trastuzumablight chain hypervariable region is shown in FIG. 2-B.

4. Comparison of Pertuzumab and Trastuzumab Light Chain Sequences andComprehensive Analysis of Light Chain and Antigen Interface Amino Acids

A comparison result of the sequences of a Pertuzumab light chain and aTrastuzumab light chain and the results of a comprehensive analysis ofantigen interface amino acids are shown in FIG. 2-C, the amino acids ofPertuzumab (P-mab) and Trastuzumab (T-mab) light chains contacting withthe antigen are shown in background black color. If the Trastuzumablight chain is designated as the common light chain, then differentialamino acids obtained by comparing the interface amino acids in thecommon light chain contacting with the antigen with those in the lightchain of Pertuzumab (P-mab) are shown in Table 3.

TABLE 3 Differential amino acids contacting with antigen from the lightchain of Pertuzumab (P-mab) and Trastuzumab (T-mab) Kabat number P-mabT-mab Kabat31 I T Kabat53 Y F Kabat56 T S Kabat94 Y T

If the Pertuzumab light chain is designated as the common light chain,then differential amino acids obtained by comparing the interface aminoacids in the common light chain contacting with the antigen with thosein the light chain of Trastuzumab (T-mab) are shown in Table 4.

TABLE 4 Differential amino acids contacting with the antigen from thelight chain of Pertuzumab (P-mab) and Trastuzumab (T-mab) Kabat numberP-mab T-mab Kabat30 S N Kabat31 I T Kabat32 G A Kabat53 Y F Kabat56 T SKabat66 G R Kabat91 Y H Kabat93 I T Kabat94 Y T

By analyzing the differential amino acids contacting with the antigenfrom the light chain of Pertuzumab (P-mab) and Trastuzumab (T-mab), thelight chain of Trastuzumab (T-mab) was selected as a framework and themutation T311 or/and T94Y were introduced to obtain a sequence of acommon light chain of a Pertuzumab and Trastuzumab bispecificantibody:CLC1-CLC4; a light chain of Pertuzumab (P-mab) was selected asa framework and the mutation of T311 or/and T94Y were introduced toobtain a sequence of a common light chain of a Pertuzumab andTrastuzumab bispecific antibody: CLC5-CLC6. Amino acid sequences of theobtained common light chain are as follows:

CLC1 (SEQ ID NO: 1) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC CLC2 (SEQ ID NO: 2)DIQMTQSPSSLSASVGDRVTITCRASQDVNIAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC CLC3 (SEQ ID NO: 3)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTYPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC CLC4 (SEQ ID NO: 4)DIQMTQSPSSLSASVGDRVTITCRASQDVNIAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTYPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC CLC5 (SEQ ID NO: 5)DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC CLC6 (SEQ ID NO: 6)DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYITPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Example 2 Preparation and Functional Verification of Antigen ProteinHER2 Variant Protein

1. Design of HER2 Variant Protein Only Binding to Pertuzumab

Robert F. Kelley' and Mark P. O'Connell published kinetic parameters ofthe binding of Trastuzumab and its corresponding mutant and HER2extracellular domain (ECD) in 1993. Wherein, H91A, R50A, W95A and Y100aAhad great influence on the binding of Trastuzumab to HER2 extracellulardomain. The team of Hyun-Soo Cho obtained crystals (PDB number: 1N8Z) ofa complex formed between a Trastuzumab Fab fragment and HER2extracellular domain (ECD) in 2003, and this result was published inNature. By analyzing the structure of the complex formed by theTrastuzumab Fab fragment and the HER2 extracellular domain (ECD) (thesequence of which is shown in SEQ ID NO:18), interface contacting aminoacids of the Trastuzumab Fab fragment and the HER2 extracellular domain(ECD) were obtained, as shown in Table 5.

H91L, R50H, W95H, Y100aH contacting amino acids were specificallyanalyzed. It was found that two combinations may significantly affectbinding of these amino acids. Combination 1: ASP570, PR0571 and PR0572,and combination 2: GLU558 and PHE573.

TABLE 5 Interface contacting amino acids of Trastuzumab Fab fragment andHER2 extracellular domain (ECD) Trastuzumab Number light chain AntigenHER2  1 ASP28L(CL1) GLU598C  2 ASN30L(CL1) PRO571C, ASP596C, GLU598C,ALA600C, CYS601C, GLN602C  3 THR31L(CL1) CYS601C, GLN602C, PRO603C  4ALA32L(CL1) PRO571C  5 TYR49L PRO603C  6 SER50L(CL2) LYS593C, PRO603C  7PHE53L(CL2) PRO603C, CYS604C, PRO605C  8 ARG66L ASP596C, GLU598C  9HIS91L(CL3) ASP570C, PRO571C, PRO572C 10 TYR92L(CL3) LYS569C, PRO571C,PRO572C, GLU598C, ALA600C 11 THR93L(CL3) ASP560C, PRO572C 12 THR94L(CL3)ASP560C, PRO572C 13 TYR33H GLU558C, PHE573C 14 ARG50H GLU558C, ASP560C,PRO572C, PHE573C 15 TYR52H GLU558C 16 TYR57H TYR532C, PRO540C, PRO557C,GLU558C 17 THR58H GLU558C 18 ARG59H GLU558C, ASP560C, GLN561C 19 TRP99HPRO572C, PHE573C 20 GLY101H ILE591C 21 ASP102H PRO579C, ILE591C 22GLY103H ASP570C, PRO579C, ILE591C, LYS593C 23 PHE104H ILE591C, LYS593C24 TYR105H ASP570C, PRO571C, PRO572C, PHE573C, VAL575C, LYS593C

It can be seen from the above result that:

combination 1: P571 and P572 interact with several critical amino acidsof the Fab. It was believed that these two amino acids locate in a loopcorner, and mutating them will influence the stability of its ownstructure.

combination 2: GLU588 forms an ion bond with and a Trastuzumab Fab heavychain ARG 50, and forms Vander Waals' forces with multiple amino acidsof the Fab heavy chain, destroying these interaction forces would blockinteractions between Trastuzumab Fab and HER2, and thus the mutationGLU558 to ALA558 was selected; Vander Waals' forces were formed betweenPHE573 and multiple amino acids of the Fab heavy chain, includingcritical amino acids ARG 508, TRP99 and TYP105, destroying theseinteraction forces would block interactions between the Fab and HER2,and thus the mutation PHE573 to ALA573 was selected.

The above analysis results are shown in FIG. 3.

2. Design of a HER2 Variant Protein Only Binding to Trastuzumab

Matthew C. Franklin published the structure of a complex formed betweenPertuzumab Fab and an HER2 extracellular domain in Cancer Cell. Thisteam also found which critical amino acids of HER2 can influence bindingto Pertuzumab Fab using the alanine scanning method. The results showthat amino acids, such as H296, S288 and L295 on the surface of the HER2protein had significant effects, and in the present application,S288A/H296A double-mutation was selected to obtain the HER2 antigen onlybinding to Trastuzumab.

Wherein, a HER2 variant protein only recognized by Trastuzumab was namedHER2m, the HER2 variant proteins only recognized by Pertuzumab werenamed HER2m2 and HER2m3. The amino acid sequences of these HER2 variantproteins are as follows:

HER2m1: (SEQ ID NO: 13) TQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGACTLVCPLANQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHS CVDLDDKGCPAEQRASPLTHER2m2: (SEQ ID NO: 14) TQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPAADQCVACAHYKDPAFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHS CVDLDDKGCPAEQRASPLTHER2m3: (SEQ ID NO: 15) TQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDAAFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHS CVDLDDKGCPAEQRASPLT

3. Modification of Commercial Mammalian Cell Expression VectorpcDNA4/myc-HisA

The commercial vector pcDNA4/myc-HisA (Invitrogen, V863-20) comprisestwo PvuII restriction enzyme recognition sites which are located at thepositions of about 1411 bp and 3160 bp, respectively. Site-directedmutagenesis was carried out on the plasmid so that a residue C at theposition 3160 bp was mutated into G, and the PvuII restriction enzymerecognition site at this position was removed while only one restrictionenzyme recognition site at the position of about 1411 bp remained, andthe new vector was named as pcDNA4m.

Primers are designed in accordance with a DNA sequence (AY623427) of acrystallizable fragment (Fc) of human immunoglobulin gamma1 (IgG1) fromNCBI as follows:

F: (SEQ ID NO: 29) AAGCTTCCCTTACCCGGATCCGAAATCCTCTGACAAAACTCAC R:(SEQ ID NO: 30) CCCGAATTCTATTTACCCGGAGACAGGGAG

wherein, HindIII and BamHI restriction enzyme recognition sites wereadded in the upstream primer for subsequent cloning, and an EcoRIrestriction enzyme recognition site was added in the downstream primer.

Full length cDNA of PBMC was used as a template for amplification toobtain genes of an Fc fragment, then the obtained gene fragments weretreated with double enzyme digestion with HindIII and BamHI from Takaracompany to be cloned into a modified vector pcDNA4m, and the accuracy ofthe constructed plasmid was verified by sequencing so as to obtain arecombinant plasmid pcDNA4m-Fc.

4. Construction of HER2 Variant Protein Eukaryotic Expression Vector

Primers are designed based on DNA sequence information (NM_004448.2) ofa HER2 protein from NCBI to clone the extracellular domain (amino acidresidues of position 1-652) of a wild type HER2 protein, and the primersused are as follows:

F: (SEQ ID NO: 31) GCCAAGCTTGCCACCATGGAGCTGGCGGCCT R: (SEQ ID NO: 32)CGCGGATCCATCGTCAGAGGGCTGGCTCTC

The primers contain upstream HindIII recognition sites and downstreamBamHI recognition sites. cDNA of a BT474 cell (purchased from ShanghaiCell Bank of Chinese Academy of Sciences) were used as a template foramplification to obtain a 1.9 kb DNA fragment, encoding theextracellular domain of HER2wt, it was cloned into a commercial T vector(pMD19-T Simple Vector purchased from Takara company) to obtain aT-Her2ECD plasmid, and the accuracy of the sequences was confirmed bysequencing.

Based on the amino acid sequences of the foregoing HER2 variant proteinHER2m1 recognized only by Trastuzumab, HER2 variant proteins HER2m2 andHER2m3 recognized only by Pertuzumab, corresponding primers weredesigned according to the mutation sites:

M1-F: (SEQ ID NO: 33) GCACCCTCGTCTGCCCCCTGGCTAACCAAGAGG M1-R:(SEQ ID NO: 34) GGGGGCAGACGAGGGTGCAAGCTCCCACGT M2-1-F: (SEQ ID NO: 35)GGACCGGCGGCTGACCA M2-1-R: (SEQ ID NO: 36) TGGTCAGCCGCCGGTCC M2-2-F:(SEQ ID NO: 37) TATAAGGACCCTGCCTTCTGCG M2-2-R: (SEQ ID NO: 38)CGCAGAAGGCAGGGTCCTTAT M3-F: (SEQ ID NO: 39) CTATAAGGACGCTGCCTTCTGCGM3-R: (SEQ ID NO: 40) CGCAGAAGGCAGCGTCCTTATAG

Using the plasmid T-Her2ECD as a template, site-specific mutagenesis wascarried out with the above primers to obtain genes of the three variantproteins (HER2m1, HER2m2, HER2m3) of HER2 extracellular domain. Then,they were treated with double enzyme digestion using HindIII and BamHIfrom Takara company and cloned into the vector pcDNA4m-Fc, the threegenes HER2m1, HER2m2 and HER2m3 were fused to the 5′ terminal of the Fcgene respectively, to obtain three new vectors named as pcDNA4m-Her2ml-Fc, pcDNA4m-Her2m2-Fc and pcDNA4m-Her2m3-Fc. These three vectors canbe used for expressing fusion proteins HER2m1-Fc, HER2m2-Fc andHER2m3-Fc in a mammalian cell.

5. Transient Expression and Purification of HER2 Variant Protein

Two days before transfection, 200 mL×3 of HEK293 (ATCC, CRL-1573™) cellsuspension was prepared for transient transfection, and an inoculationdensity was 0.8×10⁶ cells/mL. Two days later, cells in the suspension tobe transfected were counted, and the cell density was determined to be3.5-4×10⁶ cells/mL, the cell suspension was centrifuged at 1000 rpm for5 min, and the supernatant was discarded. Cells were resuspended with 40mL×3 fresh Freestyle293 culture medium and then centrifuged again at1000 rpm for 5 min, and the supernatant was discarded. 293 cells weresuspended again with 200 mL×3 Freestyle293 culture medium. 200 g of eachof th expression vectors of the three HER2 variant proteins obtained inthe examples 2-4 were diluted using 2 mL Freestyle293 culture medium,respectively. Subsequently, 1.5 mL Polyethylenimine was diluted using 5mL Freestyle293 culture medium, and a PEI solution required fortransformation was prepared. 2 mL PEI solution was respectively addedinto 2 mL diluted expression plasmids, fully mixed, and kept at roomtemperature for 5 min. Three parts of plasmid/PEI mixtures wererespectively added into three parts of 200 mL cell suspensions, andcultured under 37° C., 10% CO₂ and 90 rpm; simultaneously, 50 g/L IGF-1was added. Four hours later, 200 mL EX293 culture medium, 2 mM Glutamineand 50 g/L IGF-1 were further added into each part of the transformedsamples, and cultured with 135 rpm. 24 hours later, 3.8 mM VPA wasadded.

After culturing for 5-6 days, three parts of 400 mL HER2 variant proteincell transient expression culture supernatant was collectedrespectively, and preliminarily purified by ProteinA affinitychromatography to obtain HER2 variant protein samples. Wherein, theexpression level of HER2m3 was very low, and the titer of the templateprotein in the cell culture supernatant of HER2m3 was less than 0.5mg/L, it was suspected that this might result from instability of thevariant protein, and thus this protein was not further purified. Theexpression levels of the obtained HER2m1 and HER2m2 variant proteinswere calculated to be about 20 mg/L after purification. The obtainedprotein samples were preliminarily examined with SDS-PAGE, and targetbands were clearly seen (see FIG. 4).

6. Detection of Specific Binding of HER2 Variant Protein to Trastuzumabor Pertuzumab with ELISA

An ELISA plate was coated with a Trastuzumab protein or a Pertuzumabprotein at 4° C. overnight, then 3% BSA solution was added, and it wasblocked at room temperature for 2 hours. The sample (HER2m1 or HER2m2protein) was labeled with biotin in advance, and then the biotinylatedproteins HER2m1-Biotin and HER2m2-Biotin were gradient-diluted in 1:4,starting from 16 μg/mL and till 0.224 ng/L, including 9 gradientstotally. The gradient-diluted biotinylated HER2 variant protein sampleswere added into the ELISA plate, and the reaction was allowed at roomtemperature for 2 hours. Then, HRP-labeled streptavidin was added, andthe reaction was allowed at room temperature for 1.5 hours, and finally,the substrate was catalyzed for color development and the results wereobtained. Affinity curves were obtained by fitting of data obtainedusing a four-parameter method.

As shown in FIG. 5, the apparent affinity of the HER2m1 protein toPertuzumab was reduced by 20 times compared to that of the HER2m1protein to Trastuzumab. Thus, it can be concluded that the variantprotein is a Trastuzumab specific antigen protein. On the other hand,the apparent affinity of the HER2m2 protein to Pertuzumab was reduced bymore than 2 orders of magnitude compared to that of the HER2m2 proteinto Trastuzumab, showing that this variant protein is a Pertuzumabspecific antigen protein.

Example 4 Replacement of the Original Light Chains of Tmab and Pmab withthe Common Light Chain and Verification of the Effects of the CommonLight Chain

1. Construction of Eukaryotic Expression Vectors of Tmab and PmabMonoclonal Antibody Carrying Common Light Chains

Corresponding encoding DNA sequences were designed using DNAworkson-line tool (http://helixweb.nih.gov/dnaworks/) in accordance withamino acid sequences (FIG. 2 and FIG. 16 in the corresponding patent) ofTrastuzumab and Pertuzumab full-length antibodies found in patentUS2009/0285837A1, and the heavy chain gene (SEQ ID NO: 16) ofTrastuzumab and the heavy chain gene (SEQ ID NO: 17) of Pertuzumab wereobtained by an artificial synthesis. In accordance with the amino acidsequences (SEQ ID NO: 1-6) of a group of common light chains obtained inexample 1, corresponding DNA sequences were designed using the DNAworkson-line tool (http://helixweb.nih.gov/dnaworks/), and the gene CLC1 (SEQID NO: 7) of the common light chain of the Pmab-Tmab bispecific antibodyand the gene CLC5 (SEQ ID NO: 11) of the common light chain of thePmab-Tmab bispecific antibody were obtained by artificial synthesis.

Then mutation primers were designed in accordance with the sequences ofCLC2-CLC6, and the sequences were as follows:

T31I-F: (SEQ ID NO: 41) GACGTGAACATTGCCGTTGC T31I-R: (SEQ ID NO: 42)GCAACGGCAATGTTCACGTC T94Y-F: (SEQ ID NO: 43) AGCACTATACTTATCCTCCAACATTCT94Y-R: (SEQ ID NO: 44) ATGTTGGAGGATAAGTATAGTGCTG Y94T-F:(SEQ ID NO: 45) TCGCCACCACTTATTGTCAG Y94T-R: (SEQ ID NO: 46)CTGACAATAAGTGGTGGCGA

Using the CLC1 gene as a template, sequences of CLC2-CLC4 (SEQ ID NO:8-SEQ ID NO: 10) were obtained by site-directed mutagenesis using thetwo pairs of primers T311 and T94Y; using CLC5 as a template, thesequence of CLC6 (SEQ ID NO: 12) was obtained by site-directedmutagenesis using the Y94T primer pair.

The synthesized Trastuzumab heavy chain genes, the Pertuzumab heavychain genes and the common light chain genes (CLC1-CLC6) wererespectively subcloned into the modified vector pcDNA4m using doubleenzyme digestion with HindIII and EcoRI from Takara company, theaccuracy of plasmid construction was verified by sequencing to obtainrecombinant plasmid DNA, namely pcDNA4m-TmabHC, pcDNA4m-PmabHC and thecommon light chain related vectors pcDNA4m-CLC1 to pcDNA4m-CLC6.

The above successfully constructed common light chain gene expressionvectors pcDNA4m-CLC1 to pcDNA4m-CLC6 were treated double enzymedigestion using Bgl II and Pvu II from Takara company. Enzyme-digestedproducts were separated and purified using 0.8% agarose electrophoresis,and about 2 kb DNA fragments containing the common gene wererespectively recovered; pcDNA4m-TmabHC was treated with double enzymedigestion using BglII and NruI to recover DNA fragments of about 6 kbcontaining TmabHC genes. pcDNA4m-PmabHC was treated with double enzymedigestion using BglII and NruI to recover DNA fragments of about 6 kbcontaining PmabHC genes. Subsequently, the DNA fragments treated byenzyme digestion were ligated, and expression elements of TmabHC orPmabHC were combined with common light chain expression elements ofvarious sequences to obtain the recombinant plasmids pcDNA4m-Tmab-CLC1,pcDNA4m-Tmab-CLC2, pcDNA4m-Tmab-CLC3, pcDNA4m-Tmab-CLC4,pcDNA4m-Tmab-CLC5, pcDNA4m-Tmab-CLC6, pcDNA4m-Pmab-CLC1,pcDNA4m-Pmab-CLC2, pcDNA4m-Pmab-CLC3, pcDNA4m-Pmab-CLC4,pcDNA4m-Pmab-CLC5 and pcDNA4m-Pmab-CLC6.

2. Transient Expression and Purification of Tmab and Pmab MonoclonalAntibodies Carrying the Common Light Chains

Two days before transfection, 50 mL×12 HEK293 (ATCC, CRL-1573™) cellsuspensions were prepared for transient transfection, and an inoculationdensity was 0.8×10⁶ cells/mL. Two days later, cells in the suspension tobe transfected were counted to and the cell density was 3.5-4×10⁶cells/mL, the cell suspension was centrifuged for 5 min at 1000 rpm, anda supernatant was discarded. Cells were resuspended with a 10 mL×12fresh Freestyle293 culture medium and then centrifuged again at 1000 rpmfor 5 min, and the supernatant was discarded. 293 cells were resuspendedwith 50 mL×12 Freestyle293 culture medium. 50 g of each of the 12 commonlight chain monoclonal antibody related expression vectors obtained inthe examples 4-1 were diluted using 0.5 mL Freestyle293 culture mediumrespectively. Subsequently, 1.5 mL Polyethylenimine was diluted using 5mL Freestyle293 culture medium, and PEI solution required fortransformation was prepared. 0.5 mL PEI solution was respectively addedinto 0.5 mL diluted expression plasmids respectively, fully mixed, andkept at room temperature for 5 min. 12 parts of plasmid/PEI mixtureswere respectively added into 12 parts of 50 mL cell suspensions to becultured under 37° C., 10% CO₂ and 90 rpm; 50 g/L IGF-1 was also added.Four hours later, 50 mL EX293 culture medium, 2 mM Glutamine and 50 g/LIGF-1 were further added in each part of the transformed samples, andcultured with 135 rpm. 24 hours later, 3.8 mM VPA was added.

After culturing for 5-6 days, 12 parts of 100 mL supernatant oftransient expression culture of common light chain monoclonal antibodycells were respectively collected, and preliminarily purified usingProteinA affinity chromatography to obtain 12 common light chainmonoclonal antibody protein samples: Tmab-CLC1 to 6 and Pmab-CLC1 to 6;the expression level of each monoclonal antibody obtained afterpurification was calculated and the result is shown in Table 6.

Protein samples obtained by one-step affinity chromatographypurification were preliminarily detected using non-reduced SDS-PAGE. Asshown in FIG. 6, two clear bands can be seen for each of the commonlight chain monoclonal antibody protein on the reduced gel, which wererespectively a light chain band between 25 kDa and 35 kDa and a heavychain band between 85 kDa and 50 kDa. The purities of the proteinsamples were examined using SE-HPLC and the results are shown in Table6.

TABLE 6 Transient expression level of common light chain monoclonalantibody and sample purity after one-step purification Common lightchain monoclonal Expression level Sample purity antibody sample (mg/L)(%) TmabCLC1 56 98.7 TmabCLC2 28 98.8 TmabCLC3 38 98.7 TmabCLC4 56 96.4TmabCLC5 25 95.2 TmabCLC6 38 96.1 PmabCLC1 48 95.7 PmabCLC2 50 96.8PmabCLC3 51 96.9 PmabCLC4 46 98.9 PmabCLC5 40 98.7 PmabCLC6 42 98.9

3. Analyzing the Affinity of Tmab and Pmab Comprising the Common LightChains with ELISA

Changes in affinity of Tratuzumab and Pertuzumab comprising the commonlight chains with their respective specific antigens were examined, andthe indirect ELISA method used was similar to that in examples 2-6.Wherein, when changes in the affinity of Tratuzumab comprising thecommon light chains were examined, the specific antigen protein HER2mwas used; and when changes in the affinity of Pertuzumab comprising thecommon light chains were examined, the specific antigen protein HER2m2was used. The affinity curves obtained were shown in FIGS. 7-8.According to EC50, when the common light chains (CLC1-CLC4) obtained bymodifying the light chains of the original Tratuzumab were used, theaffinity of Tratuzumab with its specific antigen HER2m was not changedin any significant way; while the affinity of Pertuzumab with itsspecific antigen HER2m2 was slightly reduced, but this change was in anacceptable range. However, when the common light chains (CLC5 and CLC6)obtained by modifying the light chains of the original Pertuzumab wereused, the affinity of Pertuzumab with its specific antigen HER2m2 wasnot changed in any significant way, but the affinity of Tratuzumab withits specific antigen HER2m1 was reduced by nearly one-fold.

Example 5 Preparation and Identification of Ptmab Bispecific Antibody

1. Transient Expression and Purification of Ptmab Bispecific Antibody

Point mutations were introduced in Fc fragment of TmabHC inpcDNA4m-Tmab-CLC1, and TmabHC was changed to TmabHC-knob (its heavychain sequence was as set forth in SEQ ID NO: 19; the mutated residueswere S354C and T366W), and pcDNA4m-Tmabknob-CLC1 was furtherconstructed; meanwhile, amino acid residues in PmabHC ofpcDNA4m-Pmab-CLC1 were mutated into PmabHC-hole (its heavy chainsequence was as set forth in SEQ ID NO: 20; the mutated residues wereY349C, T366S, L368A and Y407V) using site-directed mutagenesis referringto patent CN102558355A, and pcDNA4m-Pmabhole-CLC1 was furtherconstructed. CN102558355A was referred to for specific mutation schemes.These two newly constructed plasmids will be used to prepare thePmab-Tmab bispecific antibody with a common light chain model, based onthe “knob-hole” model.

2. Transient Expression and Purification of Ptmab Bispecific Antibody

Two days before transfection, 600 mL of HEK293 (ATCC, CRL-1573™) cellsuspension was prepared for transient transfection, and an inoculationdensity was 0.8×10⁶ cells/mL. Two days later, cells in the suspension tobe transfected were counted, and the cell density was 3.5-4×10⁶cells/mL, the cell suspension was centrifuged at 1000 rpm for 5 min, andthe supernatant was discarded. Cells were resuspended with 100 mL freshFreestyle293 culture medium and then centrifuged again at 1000 rpm for 5min, and the supernatant was discarded. 293 cells were resuspended witha 600 mL Freestyle293 culture medium. 300 g of each ofpcDNA4m-Tmabknob-CLC1 and pcDNA4M-Pmabhole-CLC1 was fully mixed, andthen diluted using 3 mL Freestyle293 culture medium respectively.Subsequently, 1.5 mL Polyethylenimine was diluted using 5 mLFreestyle293 culture medium, and PEI solution required fortransformation was prepared. 3 mL PEI solution was added in 3 mL dilutedmixed plasmids, fully mixed, and kept at room temperature for 5 min. Theplasmid/PEI mixture was added in 600 mL cell suspensions, and culturedunder 37° C., 10% CO₂ and 90 rpm; 50 g/L IGF-1 was added as well. Fourhours later, 600 mL EX293 culture medium, 2 mM Glutamine and 50 g/LIGF-1 were added in the transformed samples again, and cultured with 135rpm. 24 hours later, 3.8 mM VPA was added.

After culturing for 6-7 days, 1200 mL supernatant of transientexpression culture of Ptmab bispecific antibody cell, and thenpreliminarily purified using ProteinA affinity chromatography,ion-exchange chromatography and molecular sieve chromatography to obtaina Ptmab bispecific protein sample named KN026. A transient expressionlevel of KN026 was as high as 80 mg/L, according to the calculationusing OD280.

The protein sample obtained after purification was preliminarilyexamined with SDS-PAGE. As shown in FIG. 9, two clear bands of the KN026bispecific antibody protein can be seen on the reduced gel, which wererespectively a light chain band between 25 kDa and 35 kaDa and a heavychain band between 85 kDa and 50 kDa. Meanwhile, under the non-reducedconditions, KN026 was shown as a single band. The purity of the proteinsample was examined using SE-HPLC and it was about 95%, the result wasshown in FIG. 10.

3. Verification of the Ability of KN026 Antibody Protein toSimultaneously Recognize the Corresponding Specific Antigens ofTrastuzumab and Pertuzumab with Bridging ELISA

An ELISA plate was coated with Trastuzumab specific antigen proteinHER2m1 at 4° C. overnight. Then, 3% BSA solution was added, and it wasblocked at room temperature for 2 hours. The sample to be tested wasgradient-diluted at 1:3, starting from 5 μg/mL to 1.06 ng/L, including 8gradients in total. The sample after gradient dilution was added intothe ELISA plate, and the reaction was allowed at room temperature for 2hours. Then, biotinylated Pertuzumab specific antigen proteinHER2m2-Biotin was added into the ELISA plate to react with the samplefor 2 hours. Subsequently, HRP labeled streptavidin was added to reactwith HER2m2-Biotin for 1.5 hours at room temperature, and finally, thesubstrate was catalyzed for color development and the results wereobtained. Affinity curves were obtained after fitting of the dataobtained, using the four-parameter method.

As shown in FIG. 11, only the bispecific antibody KN026 capable ofsimultaneously recognizing two antigens can provide an affinity curve,while for Trastuzumab or Pertuzumab capable of specifically recognizingonly one antigen, clearly developed color could not be seen even at thehighest concentration.

Example 6 Preparation and Identification of Ptmab Antibody Mixture

1. Transient Expression and Purification of Ptmab Antibody Mixture

Point mutations were introduced in Fc fragment of TmabHC inpcDNA4m-Tmab-CLC1, and TmabHC was changed to TmabHC-mix1 (wherein theheavy chain sequence was as set forth in SEQ ID NO: 21), andpcDNA4m-Tmabmix1-CLC1 was further constructed, Example 1 of patentCN103388013A was referred to for specific construction steps. Based onthe “charge repulsion” mixture model while using the common light chainmodel, these two newly constructed plasmids were used to prepare a Pmaband Tmab antibody mixture capable of being expressed in a singlerecombinant cell strain.

2. Transient Expression and Purification of Pmab and Tmab AntibodyMixture

Two days before transfection, 600 mL HEK293 (ATCC, CRL-1573™) cellsuspension was prepared for transient transfection, and an inoculumdensity was 0.8×10⁶ cells/mL. Two days later, cells in the suspension tobe transfected were counted, and the cell density was 3.5-4×10⁶cells/mL, the cell suspension was centrifuged at 1000 rpm for 5 min, andthe supernatant was discarded. Cells were resuspended with 100 mL freshFreestyle293 culture medium and then centrifuged again at 1000 rpm for 5min, and the supernatant was discarded. 293 cells were resuspended in600 mL Freestyle293 culture medium. 300 g of each ofpcDNA4m-Tmabkmix1-CLC1 and pcDNA4m-Pmab-CLC1 (wherein the heavy chainsequence was as set forth in SEQ ID NO: 22) were fully mixed, and thendiluted using 3 mL Freestyle293 culture medium. Subsequently, 1.5 mLPolyethylenimine was diluted using 5 mL Freestyle293 culture medium, andPEI solution required for transformation was prepared. 3 mL PEI solutionwas added into 3 mL diluted mixed plasmid, fully mixed, and kept at roomtemperature for 5 min. The plasmid/PEI mixture was added in 600 mL cellsuspension, and cultured under 37° C., 10% CO₂ and 90 rpm; meanwhile, 50g/L IGF-1 was added. Four hours later, 600 mL EX293 culture medium, 2 mMGlutamine and 50 g/L IGF-1 were added in the transformed samples again,and cultured with 135 rpm. 24 hours later, 3.8 mM VPA was added.

After culturing for 6-7 days, 1200 mL supernatant of transientexpression culture of Ptmab and Tmab antibody mixture cell wascollected, and then preliminarily purified using ProteinA affinitychromatography to obtain a Ptmab and Tmab antibody mixture proteinsample, named as KN010. The transient expression level of KNOW wascalculated to be as high as 100 mg/L, according to the OD280measurement.

The protein sample obtained by one-step affinity chromatographypurification was preliminarily examined with SDS-PAGE. As shown in FIG.12, two clear bands of the common light chain monoclonal antibodymixture protein product can be seen on the reduced gel, which were alight chain band between 25 kDa and 35 kDa and a heavy chain bandbetween 85 kDa and 50 kDa. Meanwhile, under the non-reduced condition,KN010 was shown as a single band. The purity of the protein sample wasexamined using SE-HPLC and the purity was about 95%, the results areshown in FIG. 13.

3. The Ability of KN01 to Recognize the Corresponding Specific Antigensof Trastuzumab and Pertuzumab was Verified Using Bridging ELISA with theSame Antigen; it was Verified that KN01 was Incapable of SimultaneouslyRecognizing this Pair of Antigens Using Bridging ELISA with DifferentAntigens.

An ELISA plate was coated with Trastuzumab specific antigen proteinHER2m1 at 4° C. overnight. Then, 3% BSA solution was added, and it wasblocked for 2 hours at room temperature. A sample to be examined wasgradient-diluted in 1:4, starting from 2.5 μg/mL until 0.61 ng/L,including 7 gradients in total. The sample to be examined after gradientdilution was added in the ELISA plate, and the reaction was allowed atroom temperature for 2 hours. Then, biotinylated Pertuzumab specificantigen protein HER2m2-Biotin or biotinylated HER2m1-Biotin was added tothe ELISA plate to react with the sample to be examined at roomtemperature for 2 hours. Subsequently, HRP-labeled streptavidin wasadded to react with HER2m2-Biotin or HER2m1-Biotin at room temperaturefor 1.5 hours, and finally the substrate was catalyzed for colordevelopment and the results were obtained. Affinity curves were obtainedby fitting of the data obtained using the four-parameter method.

An ELISA plate was coated with Perstuzumab specific antigen proteinHER2m2 at 4° C. overnight. Then, 3% BSA solution was added, and it wasblocked at room temperature for 2 hours. A sample to be examined wasgradient-diluted in 1:4, starting from 2.5 μg/mL until 0.61 ng/L,including 7 gradients in total. The sample to be examined after gradientdilution was added in the ELISA plate, and kept at room temperature for2 hours. Then, biotinylated Pertuzumab specific antigen proteinHER2m2-Biotin was added in the ELISA plate to react with the sample tobe examined at room temperature for 2 hours. Subsequently, HRP-labeledstreptavidin was added to react with HER2m2-Biotin at room temperaturefor 1.5 hours, and finally the substrate was catalyzed for colordevelopment and the results were obtained. Affinity curves were obtainedby fitting of the data obtained using the four-parameter method.

As shown in FIG. 14, it was proved in the Bridging ELISA with the sameantigen that the two arms of KN010 protein were capable ofsimultaneously recognizing the antigen of Trastuzumab, or simultaneouslyrecognizing the antigen of Pertuzumab. It indicates that the KN01protein at least comprises two antibodies capable of recognizingdifferent antigen targets. However, color development was not observedusing Bridging ELISA with different antigens, proving that the two armsof KNOW are incapable of simultaneously recognizing these two antigens,meaning that KN010 does not contain a Ptmab heterodimer component.

Example 7 Binding Activity of Ptmab Bispecific Antibody to Cell SurfaceHER2 Protein

1. Binding Activity of Ptmab Bispecific Antibody to Human Breast CancerBT474 Cell Surface HER2 Protein

Binding of HER2 over-expression breast cancer cell BT474 with HER2antibodies (such as the Ptmab bispecific antibody, Pertuzumab andTrastuzumab) was examined using flow cytometry, and concentrationdependency of the binding effects was also examined.

After digestion, BT474 cells were resuspended in 5% BSA/PBS, and 3×10⁵cells/tube were added into each 1.5 mL centrifuge tube; the samples tobe examined were diluted by three-fold, starting from 100 μg/mL till0.001694 μg/mL, including 11 concentrations in total. The samples wereallowed to interact with the cells, then FITC-rabbit anti-human IgG wasadded to test binding of the antibodies to the cells, and the meanfluorescence intensity (MFI) was recorded using flow cytometry. Curvesof MFI plotted against Logarithm values of antibody concentrations weremade, and concentration dependency curves revealing binding of theantibodies to BT474 cells were obtained by fitting using thefour-parameter method. As shown in FIG. 15, the Ptmab bispecificantibody (KN026) as well as Pertuzumab and Trastuzumab all clearly boundto BT474, and this effect was concentration dependent. As can be seenfrom EC50 of the binding curves, the affinity of KN026 to the BT474 cellsurface HER2 protein was close to that of Trastuzumab.

2. Binding of Ptmab Bispecific Antibody to Human Gastric Cancer N-87Cell Surface HER2 Protein

Binding of HER2 over-expression gastric cancer cell N-87 with HER2antibodies (such as a Ptmab bispecific antibody KN026, Pertuzumab andTrastuzumab) was examined using flow cytometry, and concentrationdependency of the binding effects was examined as well.

After digestion, N-87 cells were resuspended in 5% BSA/PBS, and 3×10⁵cells/tube were added into each 1.5 mL centrifuge tube; the samples tobe examined were diluted by two-fold, starting from 40 μg/mL till0.009766 μg/mL, including 13 concentrations in total. The samples wereallowed to react with cells, then FITC-rabbit anti-human human IgG wasadded to detect the antibodies bound to the cells, and a meanfluorescence intensity (MFI) was measured using flow cytometry. Curvesof MFI plotted against logarithm values of antibody concentrations weremade, and concentration dependency curves for the binding of theantibodies to BT474 cells were obtained by fitting using thefour-parameter method. As shown in FIG. 16, the Ptmab bispecificantibody (KN026) as well as Pertuzumab and Trastuzumab all clearly boundto N-87, and the binding effect was concentration dependent. As can beseen from EC50 of the binding curves, the affinity of KN026 to the N-87cell surface HER2 protein was slightly lower than that of Trastuzumaband Pertuzumab.

Example 8 Binding of Pmab and Tmab Antibody Mixture to Cell Surface Her2Protein

Binding Pmab and Tmab Antibody Mixture to Human Breast Cancer BT474 CellSurface HER2 Protein

Binding of HER2 over-expression breast cancer cell BT474 with HER2antibodies (such as a mixture of Pmab and Tmab antibody, Pertuzumab andTrastuzumab) was examined using flow cytometry, and concentrationdependency of the binding effects was also examined.

After digestion, BT474 cells were resuspended in 5% BSA/PBS, and 15×10⁶cells/tube were added in each 1.5 mL centrifuge tube; the samples to beexamined were diluted by three-fold, starting from 1000 μg/mL until0.01694 μg/mL, including 11 concentrations in total. The samples wereallowed to react with the cells, then FITC-rabbit anti-human was addedto detect the antibodies bound to the cells, and a mean fluorescenceintensity (MFI) was read using flow cytometry. Curves of MFI plottedagainst logarithm values of antibody concentrations were made, andconcentration dependency curves of binding of the antibodies to theBT474 cells were obtained by fitting using the four-parameter method. Asshown in FIG. 17, the mixture of Pmab and Tmab antibody (KN010) as wellas Pertuzumab and Trastuzumab all clearly bound to BT474, and thebinding effect was concentration dependent. As can be seen from EC50 ofthe curves, the affinity of KN01 to the BT474 cell surface HER2 proteinis in between those of Trastuzumab and Pertuzumab.

Example 9 Inhibition of Ptmab Bispecific Antibody on Cancer CellProliferation

1. Inhibition of Ptmab Bispecific Antibody on Human Breast Cancer BT474Cell Proliferation

The CKK-8 method was used to examine changes in the proliferation ofHER2 over-expression breast cancer cell BT474 in the presence of HER2antibodies (such as a Ptmab bispecific antibody, Pertuzumab andTrastuzumab), thereby comparing and evaluating the inhibition effect ofthe Ptmab bispecific antibody on BT474 cancer cell proliferation.

BT474 cells were adherent-cultured in a 96-well plate at a density of10000 cells/well and at 37° C. for 16 h. Samples of variousconcentrations were prepared respectively using an assay medium (DMEMculture medium, supplemented with 1% fetal calf serum): a maximumconcentration was 10 μg/ml-0.0015 g/m, and diluted by 3-fold, including9 concentrations in total. 1501 samples were added in each cell well,and 72 h later, cell vitality was measured with a CKK-8 kit (DOJNDO).The cell vitality was plotted against logarithm values of sampleconcentrations, and cell killing activity curves of the sample (Ptmabbispecific antibody KN026) and a reference (Trastuzumab andTrastuzumab+Pertuzumab combination) were obtained by fitting using thefour-parameter method.

As shown in FIG. 18, KN026 and Trastuzumab as well asTrastuzumab+Pertuzumab combination all have a clear killing effect onBT474, and this killing effect was concentration dependent. However, athigh concentration, the inhibition effect of the Ptmab bispecificantibody KN026 on BT474 cells was clearly superior to that ofTrastuzumab alone or the combination of Pertuzumab and Trastuzumab.

2. Inhibition of Ptmab Bispecific Antibody on Human Gastric Cancer N-87Cell Proliferation

Changes in proliferation of HER2 over-expression gastric cancer cellsN-87 in the presence of HER2 antibodies (such as a Ptmab bispecificantibody, Pertuzumab and Trastuzumab) were observed using a MTT method,thereby comparing and evaluating the inhibition effect of the Ptmabbispecific antibody on N-87 cancer cell proliferation.

N-87 cells were adherent cultured in a 96-well plate at a density of10000 cells/well, at 37° C. for 16 h. Samples of various concentrationswere prepared respectively using an assay medium (RPMI-1640 medium,supplemented with 1% fetal calf serum): a maximum concentration was 10μg/ml-0.0015 g/m, and diluted by three-fold, including 9 concentrationsin total. 150 μl samples were added in each cell well, and 72 h later,cell vitality was measured with a CKK-8 kit (DOJNDO). The cell vitalityobtained was plotted against the logarithm values of sampleconcentrations, and cell killing curves of the sample (Ptmab bispecificantibody) and the reference (Trastuzumab and Trastuzumab+Pertuzumabcombination) were obtained by fitting using the four-parameter method.

As shown in FIG. 19, KN026 and Trastuzumab as well asTrastuzumab+Pertuzumab combination all have a clear killing effect onN-87, and this killing effect was concentration dependent. However, athigh concentration, the inhibition effect of the bispecific antibody onN-87 is clearly superior to that of Trastuzumab alone or the combinationof Pertuzumab and Trastuzumab.

Example 10 Evaluation of the Thermal Stability of Ptmab BispecificAntibody

1. Measuring the Tm Value of Ptmab Bispecific Antibody

Tm values of a Ptmab bispecific antibody KN026 and a reference antibody(Trastuzumab was used as a reference sample herein) were measured by DSC(differential scanning calorimeter) method, and accordingly, the thermalstability of the Ptmab bispecific antibody was determined preliminarily.

The sample proteins were prepared at a concentration of 2 mg/mL in a1×PBS buffer (pH7.4). The specific heat capacities (Cp) of the samplesor blank buffer were scanned at a rate of 60° C./hr, starting from 10°C. The results of the corresponding buffer were deducted from theresults obtained for the samples, the obtained Cp values were plottedagainst the temperature, wherein, the temperature corresponding to thepeak value with clear increase of Cp is the Tm of the sample.

As shown in FIG. 20, similar to a traditional antibody, both the Ptmabbispecific antibody KN026 and the Trastuzumab reference sample clearlyshowed two Tm values, including a CH2 disassemble temperature at about60° C. and a CH3 disassemble temperature at about 80° C. Meanwhile, itcan be seen that for the Tm value at about 60° C., there was nosignificant difference between the bispecific antibody and theTrastuzumab reference; for the Tm value at about 80° C., the bispecificantibody is slightly lower but is still higher than that at 80° C., andthe difference to that of the reference was not significant, thus, shallnot be considered to affect the thermal stability of the antibody.

Example 11 Pharmacokinetic Experiment in Mice Using Ptmab BispecificAntibody

1. Examining the Metabolism of Ptmab Bispecific Antibodies in Mice

6 to 7-week old mice were selected, and divided into two groupsrandomly. The experiment group was intraperitoneally injected with 10mg/kg Ptmab bispecific antibody KN026, and the reference group wasintraperitoneally injected with 10 mg/kg of Trastuzumab referencesample. Each group was divided into three sub-groups, and blood sampleswere taken from four animals in each sub-group at different time points.During the experiment, at each time point (5 min-96 h), 0.2 ml of bloodwas taken from the orbital venous plexus of each animal; at the endpoint (192 h-576 h), blood samples were taken from the postcava aftereuthanasia of the mice with isoflurane inhalation anesthesia. After theblood samples were collected, serums were separated and temporarilystored at −80° C.

For the serum samples, blood concentration of the drugs was examinedusing Tmab and Ptmab specific ELISA, the detected amount of theantibodies in the serums was plotted against the sampling time to obtainpharmacokinetic curves of the bispecific antibody (KN026) and thereference antibody (Trastuzumab) (see FIG. 21), and correspondingPharmacokinetic parameters were further calculated Tables 7a) and 7b).As can be seen, the half-life of the Ptmab bispecific antibody (KN026)in mice was slightly lower than that of Trastuzumab, but was stilllonger than 10 days, and similar to the half-life of most monoclonalantibodies in mice, thus, it can be concluded that the stability ofPtmab in mice is similar to that of conventional monoclonal antibodies.

TABLE 7a Pharmacokinetic parameters of KN026 and Trastuzumab t_(1/2)T_(max) C_(max) AUC_(last) AUC_(INF)_obs Sample h h ug/ml h*mg/mlh*mg/ml Ref. 288.15 24 95.40 25.57 33.51 KN026 259.23  8 83.63 18.4823.33

TABLE 7b Pharmacokinetic parameters of KN026 and Trastuzumab t_(1/2)V_(d) C₁ MRT Sample h ml/kg ml/h/kg h Ref. 288.15 124.05 0.30 208.55KN026 259.23 160.29 0.43 203.23

Example 12 Pharmacodynamic Effects of Ptmab Bispecific Antibody onNude-Mouse Xenograft Model of Human Ovarian Cancer SKOV3

Balb/c nude mice were subcutaneously vaccinated with human ovariancancer SKOV3 cells at a dosage of 5×10⁶ cells+50% matrigel/mouse, andtumor-bearing mice were grouped randomly with 6 mice in each group (halfof them was males and the other half was females). When the tumor wasgrown to a volume of about 100-150 mm³, a tumor-inhibiting drug wasinjected by intraperitoneal administration twice each week for twoconsecutive weeks. The size of the tumor was measured twice each week.20 mg/kg of Ptmab bispecific antibody KN026 was administered to theexperiment group each time, 20 mg/kg of Trastuzumab reference sample orPertuzumab reference sample was administered to the reference group eachtime, and the same volume of PBS buffer was administered to a blankcontrol group each time.

As shown in FIG. 22, comparing to the blank control group, both theexperiment group and the reference group showed some tumor inhibitioneffect on the SKOV3 nude-mouse xenograft model, wherein the Ptmabbispecific antibody showed a stronger tumor inhibition effect than thatof the Trastuzumab reference sample alone or the Pertuzumab referencesample alone.

Example 13 Pharmacodynamic Effect of Ptmab Bispecific Antibody on HumanGastric Cancer N-87 Nude-Mouse Xenograft Model

Balb/c nude mice were subcutaneously inoculated with human gastriccancer N-87 cells at a dosage of 4×10⁶ cells/mouse, and tumor-bearingmice were grouped randomly with 6 mice in each group (half of them weremales and the other half were females). When the tumor grew to a volumeof about 100-130 mm³, a tumor-inhibiting drug was injected by IPadministration twice per week for 4-5 consecutive weeks. The size oftumor was measured twice per week.

The experiment group was administered each time with 5 mg/kg of PTmabbispecific antibody KN026, the reference group was administered eachtime with 5 mg/kg of Trastuzumab reference sample or Pertuzumabreference sample, and a blank control group was administered with thesame volume of PBS buffer.

As shown in FIG. 23, comparing to the blank control group, both theexperiment group and the reference group showed some tumor inhibitioneffect on the N-87 nude-mouse xenograft model, wherein the Ptmabbispecific antibody showed a tumor inhibition effect clearly superior tothat of the Trastuzumab reference sample alone or the Pertuzumabreference sample alone.

Example 14 Pharmacodynamic Effect of Ptmab Bispecific Antibody on HumanGastric Cancer N-87 Nude-Mouse Xenograft Model

Balb/c nude mice were subcutaneously inoculated with human gastriccancer N-87 cells at a dosage of 4×10⁶ cells/mouse, and tumor-bearingmice were grouped randomly with 6 mice in each group (half of them weremales and the other half were females). When the tumor grew to a volumeof about 100-120 mm³, a tumor-inhibiting drug was injected by IPadministration twice each week for three consecutive weeks. The size oftumor was measured twice each week.

2.5 mg/kg of PTmab bispecific antibody KN026 was administered to theexperiment group, 2.5 mg/kg of the combination of Trastuzumab referencesample and Pertuzumab reference sample was administered to the referencegroup each time, and the same volume of PBS buffer was administered to ablank control group each time.

As shown in FIG. 24, comparing to the blank control group, both theexperiment group and the reference group showed some tumor inhibitioneffect on the N-87 nude-mouse xenograft model, wherein the PTmabbispecific antibody showed a stronger tumor inhibition effect than thecombination of the Trastuzumab reference sample and Pertuzumab referencesample administered in an equal mole.

Example 15 Dosage Dependency of PTmab Bispecific Antibody on HER2Under-Expression Human Non-Small Cell Lung Cancer NCI-H522 MouseXenograft Model

NOD/SCID immunodeficiency mice were subcutaneously inoculated with themixture of non-small cell lung cancer NCI-H522 cells and matrigel (theratio of the cells to the matrigel was 1:1) to establish the model, theinoculation dosage was 5×10⁶ cells/mouse, and the tumor-bearing micewere grouped randomly with 6 mice in each group (half of them were malesand the other half were females). When the tumor grew to a volume ofabout 100 mm³, a tumor-inhibiting drug was injected. The day on which IP(intraperitoneal) administration was performed for the first time waslabeled as day 0. Then, IP administration was performed once each weekfor 7 consecutive weeks, and the concentration of the drugs administeredwas reduced by half (comparing to the concentration used in the firstadministration). The size of the tumor was measured twice every week.

The experiment group was divided into three sub-groups which wereadministered with PTmab bispecific antibody KN026 according to thefollowing dosages respectively: 30 mg/kg was administered for the firsttime, and subsequently, 15 mg/kg was administered each time each week;10 mg/kg was administered for the first time, and subsequently, 5 mg/kgwas administered each time each week; 3 mg/kg was administered for thefirst time, and subsequently, 1.5 mg/kg was administered each time eachweek. The blank control group was administered with the same volume ofPBS buffer each time.

As shown in FIG. 27, comparing to the blank control group, the threeexperiment groups showed clear tumor inhibiting effects on the NCI-H522mice xenograft model, and this effect was dosage dependent.

Example 16 Pharmacodynamic Effect of PTmab Bispecific Antibody on HER2Under-Expression Non-Small Cell Lung Cancer NCI-H522 Mouse XenograftModel

NOD/SCID immunodeficiency mice were subcutaneously inoculated with themixture of non-small cell lung cancer NCI-H522 cells and matrigel (theratio of the cells to the matrigel was 1:1) to establish a model, theinoculation dosage was 5×10⁶ cells/mouse, and the tumor-bearing micewere grouped randomly with 6 mice in each group (half of them were malesand the other half were females). When the tumor grew to a volume ofabout 100 mm³, a tumor-inhibiting drug was injected. The day on which IP(intraperitoneal) administration was performed for the first time waslabeled as day 0, and the dosage administered was 5 mg/kg. Then, IPadministration was performed once per week and the concentration of thedrugs administered was half of that administered in the first time,namely 2.5 mg/kg. IP administration was performed for 7 consecutiveweeks. The size of the tumor was measured twice per week.

The experiment group was administered with PTmab bispecific antibodyKN026, the reference group was administered with the combination of aTrastuzumab reference sample and a Pertuzumab reference sample, eachdrug was administered according to the dosage mentioned above, namely 5mgP+5 mgT/kg was administered for the first time, subsequently, 2.5mgP+2.5 mgT/kg was administered each time each week, and the blankcontrol group was administered with the same volume of PBS buffer eachtime.

As shown in FIG. 28, comparing to the blank control group, both theexperiment group and the reference group showed clear tumor inhibitingeffect on the NCI-H522 mice xenograft model. Meanwhile, the Ptmabbispecific antibody administered alone had an inhibiting effectcomparable to that of the Trastuzumab reference sample and thePertuzumab reference sample combination.

Although specific embodiments of the present application have beendescribed in detail, it will be understood by those skilled in the artthat according to all of the teachings publicly known in the art,various modifications and substitutions of those details may be made,which will be within the scope of the present application. The scopes ofthe present application are defined by the accompanying claims and anyequivalents thereof.

What is claimed is: 1.-45. (canceled)
 46. A method for preparing abispecific antibody, wherein said bispecific antibody comprises a commonlight chain and a heavy chain of two monoclonal antibodies, said methodcomprising a following step of: obtaining a sequence of said commonlight chain capable of assembling with said heavy chain of twomonoclonal antibodies respectively, wherein said common light chain is alight chain of one of the two monoclonal antibodies or a mutant of thelight chain of one of the two monoclonal antibodies.
 47. The method ofclaim 46, wherein the preparing method further comprises the followingstep of: obtaining a sequence of said heavy chain of two monoclonalantibodies, respectively, and modifying an Fc fragment of said heavychain to facilitate formation of a heterodimer protein.
 48. The methodof claim 47, wherein the preparing method further comprises thefollowing step of: constructing an expression vector comprising thesequence of said common light chain and the sequence of said heavy chainof the two monoclonal antibodies, respectively; and introducing saidexpression vector into the same host cell, and inducing expression toobtain said bispecific antibody.
 49. The method of claim 46, wherein avariable region of said common light chain comprises a sequence selectedfrom those as set forth in amino acid positions 1-107 of SEQ ID NO:1˜SEQ ID NO:
 6. 50. The method of claim 46, wherein a constant region ofsaid common light chain comprises a sequence as set forth in amino acidpositions 108-214 of SEQ ID NO: 1˜SEQ ID NO:
 6. 51. The method of claim46, wherein said heavy chain comprises a sequence as set forth in SEQ IDNO: 19 or SEQ ID NO: 20, respectively.
 52. The method of claim 46,wherein the preparing method further comprises the following steps of:determining an interface amino acid in a light chain variable region ofthe two monoclonal antibodies contacting with their respective antigensor antigen epitopes; designating a light chain variable region of one ofthe two monoclonal antibodies as a candidate common light chain variableregion, comparing the interface amino acid in said candidate commonlight chain variable region with those in the light chain variableregion of the other one of the two monoclonal antibodies when contactingwith the antigen or antigen epitopes directed to by said one of the twomonoclonal antibodies, to identify a differential interface amino acid;and selecting a light chain variable region comprising fewer number ofthe differential interface amino acid as a common light chain variableregion.
 53. The method of claim 52, wherein the preparing method furthercomprises the following step of: mutating the common light chainvariable region to obtain a common light chain variable region.
 54. Themethod of claim 46, wherein said bispecific antibody comprises a commonlight chain comprising a sequence selected from those as set forth inSEQ ID NO: 1˜SEQ ID NO: 6 and two heavy chains comprising a sequence asset forth in SEQ ID NO: 19 or SEQ ID NO: 20, respectively.
 55. Abispecific antibody, wherein said bispecific antibody comprises a commonlight chain and a heavy chain of two monoclonal antibodies, wherein saidcommon light chain is a light chain of one of the two monoclonalantibodies or a mutant of the light chain of one of the two monoclonalantibodies.
 56. The bispecific antibody of claim 55, wherein said commonlight chain capable of assembling with said heavy chain of twomonoclonal antibodies respectively.
 57. The bispecific antibody of claim55, wherein a variable region of said common light chain comprises asequence as set forth in amino acid positions 1-107 of SEQ ID NO: 1 orSEQ ID NO:
 5. 58. The bispecific antibody of claim 55, wherein aconstant region of said common light chain comprises a sequence as setforth in amino acid positions 108-214 of SEQ ID NO: 1 or SEQ ID NO: 5.59. The bispecific antibody of claim 55, wherein a variable region ofsaid heavy chains comprises a sequence as set forth in SEQ ID NO: 23 orSEQ ID NO: 24, respectively.
 60. The bispecific antibody of claim 55,wherein a Fc fragment of said heavy chains comprises a sequence as setforth in SEQ ID NO: 25 or SEQ ID NO: 26, respectively.
 61. Thebispecific antibody of claim 55, wherein said bispecific antibodycomprises a common light chain comprising a sequence as set forth in SEQID NO: 1 or SEQ ID NO: 5 and two heavy chains comprising a sequence asset forth in SEQ ID NO: 19 or SEQ ID NO: 20, respectively.
 62. A methodfor diagnosing, preventing and/or treating HER2 positive tumor,comprising a step of administrating to a subject in need thereof adiagnosis, prevention or treatment effective amount of a bispecificantibody or antigen binding portion thereof of claim
 10. 63. A methodfor testing whether or not an antibody or antigen binding portionthereof, which is derived from two monoclonal antibodies or antigenbinding portions thereof against different antigen epitopes, is abispecific antibody or antigen binding portion thereof, wherein the twomonoclonal antibodies or the antigen binding parts thereof are amonoclonal antibody A or a monoclonal antibody B respectively, thetesting method comprising the following steps of: a. preparing aspecific antigen 1 capable of binding the monoclonal antibody A but notthe monoclonal antibody B, wherein, when comparing to a sequence of awild type HER2 protein extracellular domain, the specific antigen 1comprises a mutation of serine at position 288 and/or a mutation ofhistidine at position 296, wherein a sequence of the wild type HER2protein extracellular domain is as set forth in SEQ ID NO: 18, b.preparing a specific antigen 2 capable of binding the monoclonalantibody B but not the monoclonal antibody A, wherein, when comparing toa sequence of a wild type HER2 protein extracellular domain, thespecific antigen 2 comprises a mutation of glutamic acid at position 558and/or a mutation of phenylalanine at position 573, c. coating an ELISAplate with the specific antigen 1 or the specific antigen 2, adding theantibody or antigen binding portion thereof to be tested, after a periodof reaction, adding the specific antigen 2 comprising a detectable labelor the specific antigen 1 comprising a detection label, after a periodof reaction, detecting the detectable label to determine the reaction aspositive or negative, d. when the reaction in the step c) is positive,the antibody or antigen binding portion thereof to be tested isdetermined as a bispecific antibody or antigen binding portion thereof.64. A method of claim 63, wherein the monoclonal antibody A isPertuzumab, and the monoclonal antibody B is Trastuzumab.
 65. A methodof claim 63, wherein the specific antigen 1 comprises a sequence as setforth in SEQ ID NO: 14 or SEQ ID NO: 15, and the specific antigen 2comprises a sequence as set forth in SEQ ID NO: 13.